CN105924521A

CN105924521A - Anti-A beta globulomer antibodies, related products thereof, methods of producing said antibodies, uses of said antibodies and methods of using said antibodies

Info

Publication number: CN105924521A
Application number: CN201510920357.6A
Authority: CN
Inventors: S.巴格霍恩; U.埃伯特; H.希伦; P.凯勒; A.斯特里滨格; B.拉布科夫斯基
Original assignee: AbbVie Deutschland GmbH and Co KG; AbbVie Inc
Current assignee: AbbVie Deutschland GmbH and Co KG; AbbVie Inc
Priority date: 2005-11-30
Filing date: 2006-11-30
Publication date: 2016-09-07
Also published as: SI1954718T1; SI1976877T1; CA2918092C; CA2918092A1; ME01826B; CN106008712A; DK2289909T3; UA95933C2; SI2289909T1; NZ629387A

Abstract

The invention relates to anti-A beta globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies. The present invention relates to anti-A beta globulomer antibodies having a binding affinity to A beta (20-42) globulomer that is greater than the binding affinity of the antibody to A beta (1-42) globulomer, antigen-binding moieties thereof, hybridomas producing said antibodies, nucleic acids encoding said antibodies, vectors comprising said nucleic acids, host cells comprising said vectors, methods of producing said antibodies, compositions comprising said antibodies, therapeutic and diagnostic uses of said antibodies and corresponding methods relating to Alzheimer's disease and other amyloidoses.

Description

Anti-A β ball dimer antibody, its Related product, the method producing described antibody, described anti- The application of body and using method

The application is in International Application Serial No. PCT/EP2006/011530 entrance that international filing date is on November 30th, 2006 State, the divisional application of application for a patent for invention of Application No. 200680052045.7.

Technical field and background technology

The present invention relates to anti-A β ball aggressiveness (globulomer) antibody, its antigen-binding portion thereof, produce the miscellaneous of described antibody Hand over tumor, the nucleic acid of encoding said antibody, comprise the carrier of described nucleic acid, comprise the host cell of described carrier, produce described anti- The method of body, comprises the compositions of described antibody, relates to the described antibody of Alzheimer and other amyloidosis Treatment and diagnostic application and corresponding method.

1907, doctor's Ai Luoyisi Alzheimer (Alois Alzheimer) described a kind of dementia first Neuropathological feature, subsequently due to its respect and named this disease (Alzheimer 1907).Alzheimer Sick (AD) be in old people modal dementia because of, in over-65s crowd, sickness rate is about 10%.With age, Incidence probability rises the most therewith.In worldwide, about 15,000,000 people suffer from this disease and life expectancy increases further, in advance Meter number of patients in coming decade can increase about 3 times.

Alzheimer (AD) from the point of view of molecule viewpoint, it is characterised in that the proteinosis thing of abnormal aggregation.Outside with regard to born of the same parents For amyloid speckle, these deposits are mainly made up of amyloid-beta-peptide silk, with regard to the neural fibre of intracellular Tau albumen Dimension is tangled for (NFTs), and amyloid-beta (A β) peptide cuts from the Proteolytic enzyme of β-amyloid precursor protein.This cutting is subject to Be referred to as α-, the impact of some proteinase synergies activity of β-and gamma-secretase.Cutting creates the specificity of many different lengths Fragment.Amyloid speckle is mainly made up of the peptide (A β 40, A β 42) of 40 or 42 amino acid lengths.Main cleaved products is A β 40；But, A β 42 has higher poisonous effect.

Be very similar in Alzheimer viewed those, Cerebral amyloid deposit and cognition lack Falling into also is the symptom of mongolism (No. 21 trisomic syndrome), and its sickness rate is about 1 example in 800 examples childbirths.

The amyloid cascade postulate of Hardy and Higgins increases the generation of A β (1-42) can cause A β plaque Main component protofibril and fibril are formed, and these fibrils are the reasons causing Alzheimer disease symptoms.Although It is related as weak between dull-witted seriousness and the A β plaque load (burden) of deposition, but this hypothesis is the most welcome up to date. In AD brain, the discovery of solubility A beta form (it is more relevant with AD symptom compared with speckle load (plaque load)) has created one and has repaiied The amyloid cascade ordered is assumed.

The active immunization using A β peptide causes speckle form minimizing and cause existing plaque components to dissolve.It is gone back simultaneously Cause the alleviation of cognitive defect in APP transgene mice model.

Also find that using the antibody for A β peptide to carry out passive immunization can reduce A β plaque load.

AN-1792 (A β (1-42) peptide being in fibril aggregation conditions) is used to carry out the IIa phase of active immunization The result of test (ELAN Corporation Plc, South San Francisco, CA, USA and Dublin, UK) thinks pin Immunization therapy to A β peptide is successful.In a subgroup of 30 patients, by MMSE and DAD assessment of indices, there is sun In the patient of the anti-A β antibody titer of property, progression of disease substantially slows down.But, owing to creating the serious side effects of meningoencephalitis, Therefore stopped this research (Bennett and Holtzman, 2005, Neurology, 64,10-12).

Meningoencephalitis has a neuroinflamation and T-cellular infiltration enters the feature of brain.Presumably, it is due to the A β (1-of injection 42) as caused by the T-cellullar immunologic response of antigen induction.After passive immunization, such immunne response is not institute's phase Hope.Up to now, relative clinical data is not yet had to utilize.But, owing to continuing 5 months weekly Preclinical study conducted in the oldest APP23 mice of the antibody accepting the N end epi-position for A β (1-42), the most Cry worry is had to be distributed about the side effect of such passive approach for immunity inoculation.With with the control animal of saline treatment Compare, these mices display increase micro-hemorrhage (microhaemorrhages) amount and seriousness (Pfeifer etc., 2002, Science, 298,1379).Also describe in Tg2576 and the PDAPP mice of very old (＞ 24 months) comparable micro-go out Blood increases (Racke etc., 2005, J Neurosci, 25,629-636；Wilcock etc. 2004, J.Neuroinflammation, 1 (1): 24；De Mattos etc., 2004, Neurobiol.Aging 25 (S2): 577).In both mices, antibody injection is led Cause the micro-hemorrhage of substantially increase.In contrast, the antibody for A β (1-42) peptide mesozone does not induce micro-hemorrhage (de Mattos etc., ibid).Induce micro-hemorrhage shortage relevant with the antibody treatment of the A β peptide not combined with the gathering of CAA form (Racke etc., J Neurosci, 25,629-636).But it is not yet clear that transgenic APP mice causes micro-hemorrhage accurate Mechanism.Presumably, cerebral amyloid angiopathy (CAA) is induced or at least exacerbates cerebral hemorrhage.CAA is present in the most each example In the brain of Alzheimer, and the case of about 20% is considered as " severe CAA ".Therefore, passive immunization should be led to Cross the antibody selecting to identify the centre of A β peptide or carboxyl terminal district and be conceived to avoid micro-hemorrhage.

WO2004/067561 describe stable A β (1-42) oligomer (A β (1-42) ball aggressiveness) and specificity for The antibody of this ball aggressiveness.Can be hydrophilic from highlighted by globose nucleus core structure with nonspecific protease digestion display A β ball aggressiveness N end plays digested (Barghorn etc., 2005, J Neurochem, 95,834-847).A β ball aggressiveness (the A β of above-mentioned N end truncate (12-42) and A β (20-42) ball aggressiveness) represent the basic structural unit of this oligomerization A β.For causing the rabbit of high antibody titer With mice active immunization, they are very effective antigen (WO2004/067561).Recently, have some to cut about N end The report present in the AD brain of short A beta form propose pathology effect that they estimate in vivo (Sergeant etc., 2003, J Neurochem, 85,1581-1591；Thal etc., 1999, J Neuropathol.Exp Neurol, 58,210-216).? In internal digestion process, some sees the such as Neprilysin of the protease in brain (neprilysin) (NEP 24.11) or insulin Digestive enzyme (IDE) may relevant with this (Selkoe, 2001, Neuron, 32,177-180).

Summary of the invention

It is an object of the present invention to provide improve in immunization therapy patient cognitive performance, the most only with in brain The antibody for A β ball aggressiveness of the sub-fraction reaction of all A β peptide.It is expected to stop the substantive disorderly of brain A β balance and produce Raw less side effect.Such as, the research of the active immunization of the A β peptide in being used in fibril aggregation conditions (uses The ELAN test of AN1792) in the minimizing suspicious in treatment of the cranial capacity that has observed that.Additionally, observed in this experiment The serious side effects existed with meningoencephalitis form.

The present invention by providing the ball aggressiveness specific antibody that the A β ball aggressiveness of clipped form is had high-affinity thus Solve this problem.These antibody not only can distinguish the A β peptide of other forms, particularly monomer and fibril, moreover it is possible to distinguishes and does not cuts The A β ball aggressiveness of short-form.

Therefore, the present invention relates to one and there is the binding affinity to A β (20-42) ball aggressiveness more than this antibody to A β (1- 42) antibody of the binding affinity of ball aggressiveness.

Additionally, the present invention relates to one there is the binding affinity to A β (20-42) ball aggressiveness more than this antibody to A β (12-42) antibody of the binding affinity of ball aggressiveness.

According to a specific embodiment, present invention is accordingly directed to that there is the combination to A β (20-42) ball aggressiveness affine Power is more than this antibody to A β (1-42) ball aggressiveness and the antibody of the binding affinity of A β (12-42) ball aggressiveness.

Term " A β (X-Y) " herein means include X and Y people's amyloid beta protein from aminoacid X position to aminoacid Y The aminoacid sequence of position, particularly aminoacid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV Its naturally occurring variant of IAT (corresponding to aminoacid 1-43 position) or any from aminoacid X position to aminoacid Y The aminoacid sequence of position, particularly those have at least one selected from A2T, H6R, D7N, A21G (" Flemish to described variant People "), E22G (" people from Arctic "), E22Q (" Dutchman "), E22K (" Italian "), D23N (" people from Iowa "), A42T The variant of the sudden change with A42V, wherein numbers relative to including X position and Y position or having at most 3 extra aminoacid and take The starting point of the A β peptide of the sequence in generation, described replacement is all without stoping the formation of ball aggressiveness, preferably from the 12nd, aminoacid or X position No matter (which numbering is higher) does not have extra amino to the 42nd, aminoacid or Y position (no matter which numbering is lower) Acid replaces, more preferably from the 20th, aminoacid or X position (no matter which numbering is higher) to the 42nd, aminoacid or Y position No matter (which numbering is lower) does not have extra aminoacid replacement, and most preferably from the 20th, aminoacid or X position No matter (which numbering is higher) does not have extra amino to the 40th, aminoacid or Y position (no matter which numbering is lower) Acid replace, " extra " aminoacid replacement at this be any one be not found in nature deviated from canonical sequence The replacement of (canonical sequence).

More specifically, term " A β (1-42) " herein means include 1 and 42 people's amyloid beta protein from aminoacid the 1st The aminoacid sequence of the 42nd, aminoacid, particularly aminoacid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA are arrived in position Its naturally occurring variant of IIGLMVGGVV IA or any, particularly those have at least one and are selected from described variant A2T, H6R, D7N, A21G (" Flemish "), E22G (" people from Arctic "), E22Q (" Dutchman "), E22K (" Italy People "), D23N (" people from Iowa "), the variant of sudden change of A42T and A42V, wherein number relative to include 1 and 42 or have to The starting point of the A β peptide of the sequence of many 3 extra aminoacid replacement, described replacement all without stop ball aggressiveness formed, preferably from The 20th, aminoacid does not has extra aminoacid replacement to the 42nd, aminoacid.Similarly, term " A β (1-40) " herein means People's amyloid beta protein including 1 and 40 from the 1st, aminoacid to the aminoacid sequence of the 40th, aminoacid, particularly amino Its naturally occurring variant of acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV or any, Particularly those have at least one selected from A2T, H6R, D7N, A21G (" Flemish "), E22G (" Arctic to described variant People "), E22Q (" Dutchman "), the variant of sudden change of E22K (" Italian ") and D23N (" people from Iowa "), wherein numbering phase For including 1 and 40 or have the starting point of the at most A β peptide of the sequence of 3 extra aminoacid replacement, described replacement is all without resistance Only ball aggressiveness is formed, and does not preferably have extra aminoacid replacement from the 20th, aminoacid to the 40th, aminoacid.

More specifically, term " A β (12-42) " herein means include 12 and 42 people's amyloid beta protein from aminoacid 12 aminoacid sequences to the 42nd, aminoacid, particularly aminoacid sequence VHHQKLVFF AEDVGSNKGA Its naturally occurring variant of IIGLMVGGVV IA or any, particularly those have at least one and are selected from described variant A21G (" Flemish "), E22G (" people from Arctic "), E22Q (" Dutchman "), E22K (" Italian "), D23N (" Chinese mugwort Watt people difficult to understand "), the variant of the sudden change of A42T and A42V, wherein number relative to including 12 and 42 or there are at most 3 extra ammonia The starting point of the A β peptide of the base substituted sequence of acid, described replacement is all without stoping the formation of ball aggressiveness, preferably from the 20th, aminoacid To the 42nd, aminoacid, not there is extra aminoacid replacement.

More specifically, term " A β (20-42) " herein means include 20 and 42 people's amyloid beta protein from aminoacid 20 aminoacid sequences to the 42nd, aminoacid, particularly aminoacid sequence F AEDVGSNKGA IIGLMVGGVV IA or appoint What its naturally occurring variant a kind of, described variant particularly those have at least one selected from A21G (" Flemish "), E22G (" people from Arctic "), E22Q (" Dutchman "), E22K (" Italian "), D23N (" people from Iowa "), A42T and The variant of the sudden change of A42V, wherein numbers relative to including 20 and 42 or have the sequence of at most 3 extra aminoacid replacement The starting point of A β peptide, described replacement is formed, preferably without any extra aminoacid replacement all without stoping ball aggressiveness.

Term " A β (X-Y) ball aggressiveness " (A β (X-Y) spherical oligomer) herein means has homogeneity and distinct thing Solvable, spherical, the non-covalent associated complex of A β (X-Y) peptide as defined above of reason characteristic.According to an aspect, A β (X-Y) ball aggressiveness is stable, non-protofibre, the widow by A β (X-Y) peptide obtainable with anionic detergent incubation Poly-set.Differing widely with monomer and fibril, these ball aggressiveness have the feature of the subunit assembling number of regulation (such as Described in WO2004/067561, such as early stage assembling form, n=4-6, " oligomer A ", and assembling form in late period, n=12- 14, " oligomer B ").This ball aggressiveness have three-dimensional spherical structure (" molten ball (molten globule) ", sees Barghorn etc., 2005, J Neurochem, 95,834-847).They can have one or more following features further:

-N terminal amino acid X-23 can cut product for mixed protease (such as thermolysin or endo protease GluC) of dwelling The ball aggressiveness of raw clipped form；

-C-end amino acid 24-Y does not dwells protease and antibody is caned close for mixed；

The clipped form of-these ball aggressiveness keeps the three-dimensional cores structure of described ball aggressiveness, at its ball aggressiveness conformation center Heart epi-position A β (20-Y) has more preferable accessibility.

According to the present invention and the binding affinity of the antibody especially for the evaluation present invention, " A β (X-Y) ball gathers term Body " particularly relate to can be obtained by the method as described in WO 2004/067561 (being herein incorporated by reference document) at this Product.

Described method includes that unfolding is natural, A β (X-Y) peptide or derivatives thereof recombinantly or synthetically；To at least partly solve folding Folded A β (X-Y) peptide or derivatives thereof is exposed to detergent, reduces decontamination continued incubation.

For unfolding peptide, hydrogen bond rupture agent such as hexafluoroisopropanol (HFIP) can be allowed to act on albumen.When operative temperature is About 20-50 DEG C, when the most about 35-40 DEG C, the action time of a few minutes, within e.g., from about 10-60 minute, it is sufficient to.Afterwards with In the mixable applicable organic solvent of water-containing buffering liquid such as dimethyl sulfoxide (DMSO), dissolve evaporation drying the most in a concentrated form Residue, produce the suspension of the peptide or derivatives thereof of at least part of unfolding, it can then be used.If necessary, Stock suspension the most such as can preserve interim a period of time at about-20 DEG C.

Alternatively, peptide or derivatives thereof can be in subacidity, the most aqueous solution, e.g., from about 10mM HCl/water solution In.After the incubative time of usual a few minutes, insoluble composition is removed by centrifugation.Under 10000g, centrifugal a few minutes are suitable 's.These method steps are preferably carried out at a temperature of room temperature (i.e. 20-30 DEG C).The supernatant obtained after Li Xin contains A β (X- Y) peptide or derivatives thereof can the most such as preserving interim a period of time at about-20 DEG C.

Below it is exposed in detergent the oligomerization relating to peptide or derivatives thereof to produce the oligomer of intermediate form (at WO Oligomer A it is referred to as) in 2004/067561.For this purpose, allow detergent act on the peptide of at least part of unfolding or it derives Thing is until enough intermediate form oligomer produce.

Ionic detergent is preferably used, particularly anionic detergent.

According to a specific embodiment, employ the detergent of logical formula (I):

R-X,

Wherein

Group R is to have 6-20, the straight or branched alkyl of preferably 10-14 carbon atom or to have 6-20 individual, preferably The straight or branched thiazolinyl of 10-14 carbon atom,

Group X is acidic-group or its salt, it is preferable that X is selected from :-COO^-M⁺、-SO₃ ^-M⁺, particularly-OSO₃ ^-M⁺, and M⁺ It is hydrogen cation or the inorganic or organic cation being preferably selected from alkali and alkaline earth metal ions cation and ammonium cation.

R is the detergent of logical formula (I) of straight chained alkyl, particularly alkane-1-base preferably wherein.Particularly preferably dodecane Base sodium sulfate (SDS).Lauric acid and oleic acid are also advantageously used.Detergent lauroyl sarcosine (lauroylsarcosin) (also referred to as sarcosyl NL-30 or) sodium salt be also particularly preferred.

In any case be all specifically dependent upon the degree of the peptide or derivatives thereof oligomerization of unfolding the action time of detergent. If according to unfolding step, the most particularly having anticipated peptide or derivatives thereof with hexafluoroisopropanol with hydrogen bond rupture agent, When operative temperature is about 20-50 DEG C and the most about 35-40 DEG C, then action time is in the range of several hours, preferably About 1-20 hour, within the most about 2-10 hour, it is sufficient to.If in the starting point less unfolding of peptide or derivatives thereof or substantially Not having unfolding, the most longer action time is expedient.If peptide or derivatives thereof has carried out pretreatment, such as, root The process of HFIP or the described direct oligomerization of peptide or derivatives thereof can be substituted, when operative temperature is about 20-50 according to aforesaid operations DEG C and when the most about 35-40 DEG C, then are sufficient to about 5-30 hour action time and the most about 10-20 hour. After incubation, insoluble composition is preferably removed by centrifugation.Under 10000g, centrifugal a few minutes are suitable.

Depend on that used detergent selects detergent concentration.If using SDS, then calculating by weight concentration is 0.01-1%, preferably 0.05-0.5%, e.g., from about 0.2% is proved to be suitable.If using lauric acid or oleic acid, slightly higher Concentration is suitable, such as, be calculated by weight to 0.05-2%, preferably 0.1-0.5%, e.g., from about 0.5%.

Decontamination should be carried out under the salinity of about physiological range.Therefore, particularly 50-500mM, preferably The NaCl concentration of 100-200mM and especially about 140mM is suitable.

Reduction and the continued incubation subsequently of decontamination relates to further oligomerization to produce the A β (X-Y) of the present invention Ball aggressiveness (is referred to as oligomer B) in WO 2004/067561.Decontamination is usually contained owing to being derived from the compositions of above-mentioned steps Agent and salinity are in physiological range, therefore facilitate and reduce decontamination and be preferably also convenient for reducing salinity. This can carry out, such as by easily with the buffer of water or more low salt concn such as by reducing detergent and salinity Tris-HCl, pH 7.3 dilutes.Have proven to dilution gfactor and be about 2-10, preferably from about 3-8, and the most about 4 is suitable. Realize reducing decontamination also by adding the material that can neutralize described decontamination.The example of these materials include can with go The material of dirty agent complexation, if stablizing the material of cell during purification and method for extracting, such as specific EO/PO block is altogether Polymers, particularly trade nameThe block copolymer of F 68.It is in around specific critical micelle concentration or is higher than Alkoxylate in the concentration range of specific critical micelle concentration and particularly ethoxylated alkylphenol are such asX series Ethoxylated tert-octylphenol, particularlyX100,3-(3-gallbladder amido propyl dimethylamino)-1-propane sulfonic acid inner saltOr alkoxylate and particularly ethoxylated sorbitan esters of fatty acids as thoseSeries , particularly20, can use comparably.

Subsequently, Incubation solution is till A β (X-Y) the ball aggressiveness of enough present invention produces.When at about 20-50 DEG C And particularly at about 35-40 DEG C during effect, action time in the range of several hours, preferably from about 10-30 hour and special It not to be sufficient to for about 15-25 hour.Then, solution can be concentrated and possible residue is removed by centrifugation.The most also one Sample, under 10000g, centrifugal a few minutes are proved to be suitable.The supernatant obtained after Li Xin contains A β (X-Y) ball of the present invention Aggressiveness.

A β (X-Y) the ball aggressiveness of the present invention can finally reclaim with a kind of method known per se, as by ultrafiltration, dialysis, Precipitation or centrifugal.

If further preferably under Denaturing, the electrophoretic separation of A β (X-Y) ball aggressiveness as by SDS-PAGE, is then produced Raw two bands (as A β (1-42) being had to the apparent molecular weight of 38/48kDa), and if the most before separation ball gather Body carries out glutaraldehyde process, then this two band merges into a band.If further preferably ball aggressiveness carries out size exclusion chromatography, divide Do not produce unimodal (as A β (1-42) the ball aggressiveness molecular weight corresponding to about 100kDa or the A β (1-for glutaraldehyde cross-linking 42) ball aggressiveness is corresponding to the molecular weight of about 60kDa).

Being initiateed by A β (1-42) peptide, A β (12-42) peptide and A β (20-42) peptide, described method is particularly suitable for obtaining A β (1- 42) ball aggressiveness, A β (12-42) ball aggressiveness and A β (20-42) ball aggressiveness.

In a particular of the present invention, wherein X is A β as defined above selected from numeral 2..24 and Y (X-Y) ball aggressiveness be those by A β (1-Y) ball aggressiveness is truncated to wherein X selected from numeral 2..24, preferably 20 or 12 and Y The obtainable antibody for more short-form as defined above, can be by obtaining by suitable Protease Treatment.Such as, can lead to Cross and A β (1-42) ball aggressiveness is carried out thermolysin Proteolytic enzyme acquisition A β (20-42) ball aggressiveness, and can be by A β (1-42) ball aggressiveness carries out endo protease GluC Proteolytic enzyme acquisition A β (12-42) ball aggressiveness.When reaching desired albumen water During solution degree, inactivating protein enzyme in the way of generally known.After the operation already described herein, the ball of resulting separation gathers Body, and if necessary, be further processed with purification step by further checking.Described process detailed Description is disclosed in WO 2004/067561, and it is herein incorporated by reference.

For the present invention, A β (1-42) ball aggressiveness A β (1-as described in embodiment hereof 1a specifically 42) ball aggressiveness；A β (20-42) ball aggressiveness A β (20-42) ball aggressiveness as described in embodiment hereof 1c specifically, with And A β (12-42) ball aggressiveness A β (12-42) ball aggressiveness as described in embodiment hereof 1d specifically.

Preferably, ball aggressiveness has the affinity to neuronal cell.Preferably, ball aggressiveness also has neuroregulation work With.

According to another aspect of the present invention, ball aggressiveness is by 11-16 A β (X-Y) peptide and most preferably individual by 12-14 A β (X-Y) peptide forms.

According to another aspect of the present invention, term " A β (X-Y) ball aggressiveness " herein means substantially by A β (X-Y) subunit The ball aggressiveness of composition, the subunit of at least 11/12 is A β (X-Y) type the most on an average, and the ball of more preferably less than 10% gathers Body comprises any non-A β (X-Y) peptide, and the content of most preferably non-A β (X-Y) peptide is less than detection threshold value.

More specifically, term " A β (1-42) ball aggressiveness " herein means substantially by A β (1-42) unit as defined above The ball aggressiveness of composition；Term " A β (12-42) ball aggressiveness " herein means and is substantially made up of A β (12-42) unit as defined above Ball aggressiveness；And term " A β (20-42) ball aggressiveness " herein means and is substantially made up of A β (20-42) unit as defined above Ball aggressiveness.

Term " A β (X-Y) the ball aggressiveness of crosslinking " be herein means and cross-linked, by crosslinking, preferably chemical crosslinking, more preferably aldehyde Preferably glutaraldehyde cross-linking ball aggressiveness composition unit, is available from the molecule of A β (X-Y) ball aggressiveness as described above.In the present invention Another aspect in, the ball aggressiveness of crosslinking substantially wherein unit is to connect at least partially by covalent bond rather than only passes through The ball aggressiveness that noncovalent interaction combines.For the present invention, A β (1-42) the ball aggressiveness of crosslinking is specifically such as this A β (1-42) oligomer of the crosslinking described in literary composition embodiment 1b.

Term " A β (X-Y) ball mer derivatives " particularly relates at this be marked by the covalently bound group being easy to detection The ball aggressiveness of note, the preferred fluorogen of described group, such as Fluorescein isothiocyanate, phycoerythrin, Victoria's multitube luminescent jellyfish (Aequorea Victoria) fluorescin, net belong to (Dictyosoma) fluorescin or their any combination or fluorescence Reactive derivative；Chromophore；Chemical luminophor, such as luciferase, preferably North America Lampyridea (Photinus pyralis) fluorescence Element enzyme, Fermi operator (Vibrio fischeri) luciferase or their any combination or chemiluminescence activity derivant；Enzyme Active group, such as peroxidase, such as horseradish peroxidase or its any enzymatic activity derivant；High electron density group, as Containing the group of heavy metal, such as the group containing gold；Hapten, hapten as derivative in phenol；Strong antigen structure, has as anticipated Antigenic peptide sequence, as having antigenic peptide sequence by the algorithm of Kolaskar and Tongaonkar is anticipated；For separately A kind of molecule fit；Chelation group, such as six histidine bases；The further spy of natural or natural derivative protein structure mediation Foreign preteins-protein-interacting, if fos/jun is to member；Magnetic group, such as ferromagnetism group；Or radioactive group, such as bag Contain¹H、¹⁴C、³²P、³⁵S or¹²⁵I or their any combination of group；Or refer to by covalently or non-covalently high-affinity phase interaction With the ball aggressiveness of institute's label, preferably covalently connects to have is easy to inactivation, multivalence chelating, degraded and the group of/precipitation of living, preferably with promoting Enter the group of vivo degradation, more preferably carry out label with ubiquitin, if the oligomer at this this label assembles in vivo, be special The most preferred；Or refer to the ball aggressiveness modified by any of above combination.Above-mentioned labelling and tag groups and for by them even The method being connected on albumen is known in the art.Labelling and/or label can be carried out before, during or after ball is multimerizing. In another aspect of the present invention, ball mer derivatives be by labelling and/or tag reactant be available from ball aggressiveness point Son.

Correspondingly, term " A β (X-Y) monomer derived thing " particularly relate at this as to labeled described by ball aggressiveness or The A beta monomers of label.

Advantageously, the antibody of the present invention is with 1x10^-6M-1x10^-12The K of M_DIn conjunction with A β (20-42) ball aggressiveness.Preferably, anti- Body combines A β (20-42) ball aggressiveness with high-affinity, such as with 1x10^-7The K of M_DOr bigger affinity, as with 3x10^-8The K of M_D Or bigger affinity, with 1x10^-8The K of M_DOr bigger affinity, as with 3x10^-9The K of M_DOr bigger affinity, with 1x10^-9The K of M_DOr bigger affinity, as with 3x10^-10The K of M_DOr bigger affinity, with 1x10^-10The K of M_DOr bigger parent And power, as with 3x10^-11The K of M_DOr bigger affinity, or with 1x10^-11The K of M_DOr bigger affinity combines.

Term " bigger affinity " herein means the degree of a kind of interaction, the most unconjugated antibody and unconjugated On one side, antibody-ball dimeric complexes is to be more beneficial for antibody-ball dimeric complexes in the balance of another side to ball aggressiveness.Equally Ground, it is poly-that term " less affinity " herein means the degree of a kind of interaction, the most unconjugated antibody and unconjugated ball Body on one side and antibody-ball dimeric complexes is to be more beneficial for unconjugated antibody and unconjugated ball gathers in the balance of another side Body.Term " bigger affinity " and term " higher affinity " are synonyms, and term " less affinity " and art Language " lower affinity " is synonym.

According to a specific embodiment, the present invention relates to 1x10^-6M-1x10^-12The K of M_DIn conjunction with A β (20-42) ball Aggressiveness, and with 10^-12The K of M_DOr less affinity combines A β (1-42) ball aggressiveness, the combination to A β (20-42) ball aggressiveness Affinity is more than the antibody of the binding affinity to A β (1-42) ball aggressiveness.

The preferably antibody of the present invention is at least 2 times to the binding affinity of A β (20-42) ball aggressiveness, such as at least 3 times or extremely Few 5 times, preferably at least 10 times, such as at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, as at least 200 times, At least 300 times or at least 500 times, and further preferably at least 1000 times, such as at least 2000 times, at least 3000 times or at least 5000 Times, more preferably at least 10000 times, such as at least 20000 times, at least 30000 or at least 50000 times, and most preferably at least 100000 times are more than this antibody binding affinity to A β (1-42) ball aggressiveness.

According to a specific embodiment, the present invention relates to 10^-12The K of M_DOr less affinity combines A β (12- 42) ball aggressiveness, binding affinity the resisting more than the binding affinity to A β (12-42) ball aggressiveness to A β (20-42) ball aggressiveness Body.

The further preferably antibody of the present invention is at least 2 times to the binding affinity of A β (20-42) ball aggressiveness, such as at least 3 times or At least 5 times, preferably at least 10 times, such as at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, such as at least 200 Times, at least 300 times or at least 500 times, and further preferably at least 1000 times, such as at least 2000 times, at least 3000 times or at least 5000 times, more preferably at least 10000 times, such as at least 20000 times, at least 30000 or at least 50000 times, and most preferably at least 100000 times are more than this antibody binding affinity to A β (12-42) ball aggressiveness.

Preferably, at least one A β ball aggressiveness as defined above of the antibodies of the present invention, and at least one A β's Aspheric dimer form has relatively smaller affinity.

With compared with at least one A β ball aggressiveness, the aspheric dimer form of at least one A β is had relatively smaller affine The antibody of the present invention of power includes having the binding affinity to A β (20-42) ball aggressiveness more than the combination to A β (1-42) monomer The antibody of affinity.Optionally, in addition or additionally preferred antibody to the binding affinity of A β (20-42) ball aggressiveness more than to A β (1-40) binding affinity of monomer.

In a preferred embodiment in accordance with this invention, antibody is more than it to A to the affinity of A β (20-42) ball aggressiveness β (1-40) and the affinity of A β (1-42) monomer.

Term " A β (X-Y) monomer " herein means the unpack format of A β (X-Y) peptide, refer preferably to one substantially non-involvement with Other A β peptide produce the form of A β (X-Y) peptide of noncovalent interaction.It is true that A β (X-Y) monomer is generally with aqueous solution shape Formula provides.In a particularly preferred embodiment of the present invention, monomer solution contains 0.05%-0.2%, more preferably from about The NH of 0.1%₄OH.In another particularly preferred embodiment of the present invention, monomer solution contains 0.05%-0.2%, The NaOH of more preferably from about 0.1%.When using (such as measuring the binding affinity of the antibody of the present invention), can be suitable Mode dilutes described solution easily.Additionally, be frequently advantageous that in latter 2 hours of its precipitation, in particularly 1 hour and outstanding It used described solution in 30 minutes.

More specifically, term " A β (1-40) monomer " herein means A β (1-40) list as described in embodiment hereof 2 Body vegetation thing, and term " A β (1-42) monomer " herein means A β (1-42) goods as described in embodiment hereof 2.

Advantageously, the antibody of the present invention combines a kind of with low-affinity or more preferably combines two kinds of monomers, most preferably with 1x10^-8The K of M_DOr less affinity, as with 3x10^-8The K of M_DOr less affinity, with 1x10^-7The K of M_DOr less parent And power, as with 3x10^-7The K of M_DOr less affinity, or with 1x10^-6The K of M_DOr less affinity, as with 3x10^-5M's K_DOr less affinity, or with 1x10^-5The K of M_DOr less affinity combines.

The particularly preferably antibody of the present invention is at least 2 times to the binding affinity of A β (20-42) ball aggressiveness, as at least 3 times or at least 5 times, preferably at least 10 times, such as at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, as extremely Few 200 times, at least 300 times or at least 500 times, and further preferably at least 1000 times, such as at least 2000 times, at least 3000 times or extremely Few 5000 times, more preferably at least 10000 times, such as at least 20000 times, at least 30000 or at least 50000 times, and the most extremely Few 100000 times more than this antibody to a kind of or more preferably binding affinity to two kinds of monomers.

With compared with at least one A β ball aggressiveness, the aspheric dimer form of at least one A β is had relatively smaller affine The antibody of the present invention of power farther includes have the binding affinity to A β (20-42) ball aggressiveness more than to A β (1-42) fibril The antibody of the binding affinity of dimension.Optionally, in addition or the additionally preferred antibody binding affinity to A β (20-42) ball aggressiveness More than the binding affinity to A β (1-40) fibril.

Term " fibril " herein means the molecule knot of the assembly of a kind of single A β (X-Y) peptide comprising noncovalent associations Structure, it shows fibrillar structure in ultramicroscope, has birefringence, and its in conjunction with Congo red the most under polarized light X-ray diffracting spectrum be intersection-beta structure.

In another aspect of the present invention, fibril is a kind of by comprising as in 0.1M HCl, in detergent not In the presence of the syndication aggregation of the suitable auto-induction of A β peptide, described gathering causes more than 24 monomers, preferably more than The obtainable molecular structure of process that the polymer of 100 monomers is formed.This is processed as it is well known in the art that.Advantageously, A β (X-Y) fibril is used as an aqueous solution.In a particularly preferred embodiment of the present invention, by by A β peptide It is dissolved in 0.1%NH₄In OH, use 20mM NaH₂PO₄, 140mM NaCl, pH 7.4 dilutes it with 1: 4, then adjusts pH to 7.4, Incubation solution 20 hours at 37 DEG C, are then centrifuged 10 minutes under 10000g and are resuspended in 20mM NaH₂PO₄, 140mM NaCl, pH 7.4 is prepared fibril aqueous solution.

Term " A β (X-Y) fibril " herein means the fibril being substantially made up of A β (X-Y) subunit, wherein average Get up at least 90% subunit be A β (X-Y) type, the subunit of more preferably at least 98% is A β (X-Y) type, and most preferably non-A β (X-Y) content of peptide is less than detection threshold value.

More specifically, term " A β (1-42) fibril " herein means the A β (1-42) as described in embodiment hereof 3 Fibril goods.

Advantageously, the antibody of the present invention combines a kind of with low-affinity or more preferably combines two kinds of fibrils, most preferably With 1x10^-8The K of M_DOr less affinity, such as 3x10^-8The K of M_DOr less affinity, with 1x10^-7The K of M_DOr less parent And power, as with 3x10^-7The K of M_DOr less affinity, or with 1x10^--⁶The K of M_DOr less affinity, as with 3x10^-5M's K_DOr less affinity, or with 1x10^-5The K of M_DOr less affinity combines.

The particularly preferably antibody of the present invention is at least 2 times to the binding affinity of A β (20-42) ball aggressiveness, as at least 3 times or at least 5 times, preferably at least 10 times, such as at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, as extremely Few 200 times, at least 300 times or at least 500 times, and further preferably at least 1000 times, such as at least 2000 times, at least 3000 times or extremely Few 5000 times, more preferably at least 10000 times, such as at least 20000 times, at least 30000 or at least 50000 times, and the most extremely Few 100000 times more than this antibody to a kind of or more preferably binding affinity to two kinds of fibrils.

According to a specific embodiment, the present invention relates to that there is the binding affinity to A β (20-42) ball aggressiveness big In it to A β (1-40) and the antibody of the binding affinity of A β (1-42) fibril.

According to a specific preferred embodiment, the present invention relates to and at least one A β ball aggressiveness, particularly A β (20-42) ball aggressiveness is compared, and has the antibody of the affinity relatively smaller to A beta monomers and fibril form.These antibody under Referred to herein as ball aggressiveness specific antibody.

The antibody of the present invention farther includes have the binding affinity to A β (20-42) ball aggressiveness more than the A β to crosslinking (1-42) ball aggressiveness, the particularly antibody to the binding affinity of A β (1-42) the ball aggressiveness of glutaraldehyde cross-linking, such as herein Antibody described in embodiment 1b.

In a particularly preferred embodiment of the present invention, the antibody binding affinity to A β (20-42) ball aggressiveness It is at least 2 times, such as at least 3 times or at least 5 times, preferably at least 10 times, such as at least 20 times, at least 30 times or at least 50 times, more excellent Select at least 100 times, such as at least 200 times, at least 300 times or at least 500 times, and further preferably at least 1000 times, such as at least 2000 Times, at least 3000 times or at least 5000 times, more preferably at least 10000 times, as at least 20000 times, at least 30000 or at least 50000 times, and most preferably at least 100000 times more than this antibody binding affinity of A β (1-42) ball aggressiveness to crosslinking.

The antibody of the present invention preferably separates, the most monoclonal and more particularly recombinate.

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7241 (5F7)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7239 (10F11)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7240 (7C6)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7242 (4B7)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7409 (6A2)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7408 (2F2)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7405 (4D10)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7809 (7E5)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7810 (10C1)。

The invention still further relates to the monoclonal antibody being available from being appointed as the hybridoma of ATCC deposit number PTA-7851 (3B10)。

The antibody of these present invention, 5F7,10F11,7C6,4B7,6A2,2F2,4D10,7E5,10C1 and 3B10, it is right to have The binding affinity of A β (20-42) ball aggressiveness is more than these antibody feature to the binding affinity of A β (1-42) ball aggressiveness.

The invention still further relates to monoclonal antibody 5F7 described with any one, 10F11,7C6,4B7,6A2,2F2,4D10, 7E5,10C1 have the antibody of similar bonding state (binding profile) with 3B10.Have and be similar to any institute The antibody of the bonding state stating monoclonal antibody should be understood to be not limited to have the binding affinity to A β (20-42) ball aggressiveness More than this antibody antibody to the binding affinity of A β (1-42) ball aggressiveness.

Have bonding state be similar to any described monoclonal antibody 5F7,10F11,7C6,4B7,6A2,2F2, The antibody of the bonding state of 4D10,7E5,10C1 and 3B10 include with monoclonal antibody 5F7,10F11,7C6,4B7,6A2,2F2, 4D10,7E5,10C1 and 3B10 combine the antibody of identical epi-position.

All monoclonal antibodies from 5F7,10F11,7C6,4B7,6A2,2F2,4D10,7E5,10C1 and 3B10 are all tied Within conjunction is included in 20-42 A β sequence context, the particularly epi-position within 20-30 A β sequence context.It is not bound to theory, Think that described epi-position is in aminoacid 20-42 region, the knot being especially between the subunit in aminoacid 20-30 region Epi-position structure, nonlinear.

The invention still further relates to can with at least one, preferably all of selected from 5F7,10F11,7C6,4B7,6A2,2F2, The antibody of the antibody competition of 4D10,7E5,10C1 and 3B10.

Term " competitive antibody " herein means multiple targeting same molecular or stably but noncovalently to connect supermolecule real The antibody of the molecule that body is the most identical, at least one of which antibody energy specificity reduces measurable combination of another kind of antibody, Preferably by spatially hinder other antibody close to its target epi-position or by the conformation in induction and/or stable target entity thus Reduce target affinity to other antibody, more preferably by combining the target epi-position very close to other antibody, overlap or with Identical epi-position, most preferably directly block by combining overlapping or identical, the most identical epi-position close to other resist The target epi-position of body.If two epi-positions they jointly enjoy their chemical constitution of a part preferably their aminoacid sequence Row, then be considered as " overlapping " at this, and if their chemical constitution preferably their aminoacid sequence be identical, It is considered as then " identical ".

Therefore, the invention still further relates to its target epi-position with at least one selected from 5F7,10F11,7C6,4B7,6A2,2F2, The antibody that the target epi-position of the antibody of 4D10,7E5,10C1 and 3B10 is overlapping, the most same.

Therefore, monoclonal antibody 5F7 described with any one, 10F11,7C6,4B7,6A2,2F2,4D10,7E5,10C1 Have with 3B10 the antibody of similar bonding state farther include to comprise any described monoclonal antibody 5F7,10F11, The antibody of at least one of antigen-binding portion thereof of 7C6,4B7,6A2,2F2,4D10,7E5,10C1 and 3B10.Preferably, institute State part and comprise any described monoclonal antibody 5F7,10F11,7C6,4B7,6A2,2F2,4D10,7E5,10C1 and 3B10 At least one complementary determining region (CDR).

Therefore, according to a further particular, the present invention relates to comprise monoclonal antibody 5F7, The aminoacid sequence of the heavy chain CDR3 of 10F11,7C6,4B7,6A2,2F2,4D10,7E5,10C1 or 3B10 and/or light chain CDR3 The antibody of aminoacid sequence.The object lesson of above-mentioned antibody include those comprise the most respectively monoclonal antibody 5F7,10F11, The aminoacid sequence of the heavy chain CDR2 of 7C6,4B7,6A2,2F2,4D10,7E5,10C1 or 3B10 and/or the amino of light chain CDR2 The antibody of acid sequence.More particularly still say, above-mentioned antibody include those comprise the most respectively monoclonal antibody 5F7,10F11,7C6, The aminoacid sequence of the heavy chain CDR1 of 4B7,6A2,2F2,4D10,7E5,10C1 or 3B10 and/or the aminoacid sequence of light chain CDR1 The antibody of row.

In an aspect, present invention is accordingly directed to comprise wherein CDR3, CDR2 and/or CDR1 district and contain monoclonal antibody The amino of heavy chain CDR3, CDR2 and/or CDR1 of 5F7,10F11,7C6,4B7,6A2,2F2,4D10,7E5,10C1 or 3B10 The antibody of the heavy chain of acid sequence.

In one aspect of the method, present invention is accordingly directed to comprise wherein CDR3, CDR2 and/or CDR1 district and contain monoclonal anti The amino of light chain CDR3, CDR2 and/or CDR1 of body 5F7,10F11,7C6,4B7,6A2,2F2,4D10,7E5,10C1 or 3B10 The antibody of the light chain of acid sequence.

Preferably, antibody comprises at least one ammonia containing the CDR:SEQ ID NO:3 selected from following aminoacid sequence Amino acid residue 99-109, SEQ of amino acid residue 50-66, SEQ ID NO:3 of base acid residue 31-35, SEQ ID NO:3 The amino acid residue of amino acid residue 55-61, SEQ ID NO:4 of amino acid residue 24-39, SEQ ID NO:4 of ID NO:4 Amino acid residue 50-66, SEQ ID NO:7 of amino acid residue 31-35, SEQ ID NO:7 of 94-102, SEQ ID NO:7 Amino acid residue 97-109, SEQ ID NO:8 amino acid residue 24-39, SEQ ID NO:8 amino acid residue 55-61, The ammonia of amino acid residue 31-35, SEQ ID NO:11 of amino acid residue 94-102, SEQ ID NO:11 of SEQ ID NO:8 The amino acid residue 24-39 of amino acid residue 98-107, SEQ ID NO:12 of base acid residue 50-65, SEQ ID NO:11, The ammonia of amino acid residue 94-102, SEQ ID NO:15 of amino acid residue 55-61, SEQ ID NO:12 of SEQ ID NO:12 The amino acid residue 99-107 of amino acid residue 50-66, SEQ ID NO:15 of base acid residue 31-35, SEQ ID NO:15, The ammonia of amino acid residue 56-62, SEQ ID NO:16 of amino acid residue 24-40, SEQ ID NO:16 of SEQ ID NO:16 The amino acid residue 50-66 of amino acid residue 31-35, SEQ ID NO:19 of base acid residue 95-103, SEQ ID NO:19, The ammonia of amino acid residue 24-39, SEQ ID NO:20 of amino acid residue 99-109, SEQ ID NO:20 of SEQ ID NO:19 The amino acid residue 31-35 of amino acid residue 94-102, SEQ ID NO:23 of base acid residue 55-61, SEQ ID NO:20, The ammonia of amino acid residue 99-109, SEQ ID NO:24 of amino acid residue 50-66, SEQ ID NO:23 of SEQ ID NO:23 The amino acid residue 94-102 of amino acid residue 55-61, SEQ ID NO:24 of base acid residue 24-39, SEQ ID NO:24, The ammonia of amino acid residue 50-65, SEQ ID NO:27 of amino acid residue 31-35, SEQ ID NO:27 of SEQ ID NO:27 The amino acid residue 55-61 of amino acid residue 24-39, SEQ ID NO:28 of base acid residue 98-101, SEQ ID NO:28, The ammonia of amino acid residue 31-35, SEQ ID NO:31 of amino acid residue 94-102, SEQ ID NO:31 of SEQ ID NO:28 The amino acid residue 24-40 of amino acid residue 99-107, SEQ ID NO:32 of base acid residue 50-66, SEQ ID NO:31, The ammonia of amino acid residue 95-103, SEQ ID NO:35 of amino acid residue 56-62, SEQ ID NO:32 of SEQ ID NO:32 The amino acid residue 99-107 of amino acid residue 50-66, SEQ ID NO:35 of base acid residue 31-35, SEQ ID NO:35, The ammonia of amino acid residue 56-62, SEQ ID NO:36 of amino acid residue 24-40, SEQ ID NO:36 of SEQ ID NO:36 The amino acid residue 50-66 of amino acid residue 31-35, SEQ ID NO:38 of base acid residue 95-103, SEQ ID NO:38 and The amino acid residue 98-109 of SEQ ID NO:38.

In a preferred embodiment, antibody comprises at least 3 CDR in the sequence of above-mentioned disclosure.More excellent Selection of land, this is chosen for 3 CDR is from selected from following variable region CDR group:

In one embodiment, the antibody of the present invention comprises at least two variable region CDR group.It is highly preferred that the two Variable region CDR group is selected from: VH 5F7 CDR group &VL 5F7 CDR group；VH 10F11 CDR group &VL 10F11 CDR group；VH 7C6 CDR group &VL 7C6 CDR group；VH 4B7 CDR group &VL 4B7 CDR group；VH 2F2 CDR group &VL 2F2 CDR group；VH 6A2 CDR group &VL 6A2 CDR group；VH 4D10 CDR group &VL 4D10 CDR group；VH 7E5 CDR group &VL 7E5 CDR group； With VH 10C1 CDR group &VL 10C1 CDR group.

In another embodiment, antibody described above comprises people acceptor framework region further.

In a preferred embodiment, antibody is CDR grafted antibody.Preferably, CDR grafted antibody comprise one or The CDRs of multiple above-mentioned disclosures.

Preferably, CDR grafted antibody comprises people acceptor framework region.

In a preferred embodiment, antibody is humanized antibody.Preferably, humanized antibody comprises one or many The CDRs of individual above-mentioned disclosure.It is highly preferred that humanized antibody comprises the CDRs of three or more above-mentioned disclosures.Most preferably, Humanized antibody comprises the CDRs of 6 above-mentioned disclosures.In a specific embodiment, CDRs is inserted people antibody variable region People acceptor framework region in.Preferably, people antibody variable region is somebody variable region altogether.It is highly preferred that people acceptor framework region exists Comprising at least one framework region amino acid at Key residues to replace, wherein Key residues is selected from the residue being adjacent to CDR；Glycosylation Site residue；Rare residue；Can be on the influential residue of A β (20-42) ball aggressiveness；Can residue influential on CDR；Specification is residual Base；Contact residues between variable region of heavy chain and variable region of light chain；Vernier (Vernier) district residue；With Chothia definition can Become the residue in overlay region between the first heavy chain framework region of heavy chain CDR1 and Kabat definition.Preferably, people acceptor framework region bag Replacing containing at least one framework region amino acid, wherein the aminoacid sequence at least 65% of framework region is same as described people's acceptor framework The sequence in district and comprise at least 70 amino acid residues identical with described people acceptor framework region.

In a further aspect, the present invention relates to comprise the antibody of heavy chain as defined above and light chain.

Preferably, antibody comprises at least one and has and be selected from: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 31, the variable region of the aminoacid sequence of SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:38. It is highly preferred that antibody comprises two variable regions, wherein said two variable regions have and are selected from: SEQ ID NO:3&SEQ ID NO: 4, SEQ ID NO:7&SEQ ID NO:8&SEQ ID NO:11&SEQ ID NO:12, SEQ ID NO:15&SEQ ID NO: 16, SEQ ID NO:19&SEQ ID NO:20, SEQ ID NO:23&SEQ ID NO:24, SEQ ID NO:27&SEQ ID NO:28, SEQ ID NO:31&SEQ ID NO:32 and the aminoacid sequence of SEQ ID NO:35&SEQ ID NO:36.

In one aspect of the method, the antibody of the present invention comprise selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and the CH of human IgG1's Ala234 Ala235 mutant constant region.Particularly, antibody comprises human constant region.More Preferably, antibody comprises the aminoacid sequence selected from SEQ ID NOs:39-42.Preferably comprise the antibody of IgGl CH.

In another embodiment, antibody is glycosylated antibody.Preferably glycosylation type behaviour glycosylation type or at this Glycosylation type produced by any eukaryotic cell particularly Chinese hamster ovary celI disclosed.

The invention still further relates to the antigen-binding portion thereof of the antibody of the present invention.Above-mentioned antigen-binding portion thereof includes but not limited to resist The Fab fragment of body, F (ab ')₂Fragment and Single-Chain Fv Fragment of Murine.More antigen-binding portion is divided into Fab ' fragment, Fv fragment and two sulfur Bonded Fv fragment.

Present invention also offers the nucleic acid of a kind of separation encoding any antibody disclosed here.Further implement Scheme provides the carrier of a kind of nucleic acid comprising separation disclosed here.Described carrier can be in particular selected from pcDNA；pTT (Durocher etc., Nucleic Acids Research 2002, Vol 30, No.2)；PTT3 (has extra polyclone position The pTT of point)；PEFBOS (Mizushima, S. and Nagata, S., (1990) Nucleic Acids Research Vol 18, No.17)；pBV；PJV and pBJ.

In one aspect of the method, with vector host cell described herein.Preferably, host cell is that protokaryon is thin Born of the same parents.It is highly preferred that host cell is escherichia coli (E.coli).In a related embodiment, host cell is that eucaryon is thin Born of the same parents.Preferably, eukaryotic cell is (as thin in mammalian cell, avian cells and insecticide selected from protist cell, zooblast Born of the same parents), plant cell and fungal cell.It is highly preferred that host cell is mammalian cell, include but not limited to CHO and COS； Or be fungal cell, such as yeast cells, such as saccharomyces cerevisiae (Saccharomyces cerevisiae)；Or be insect cell such as Sf9。

Another aspect of the present invention provides a kind of method of antibody producing the present invention, be included in be suitable for producing anti- Under conditions of body, cultivate any host cell described herein or hybridoma in the medium.Another embodiment Provide a kind of by the obtainable antibody of methods disclosed herein.

The antibody of the present invention can obtain in a manner known per se.

All containing by the bone-marrow-derived lymphocyte of the hundreds of kind of antibody library formed in billions of kinds of different antibodies specificitys is to feed A part for breast animal immune system.Normal immunological response to specific antigen means described in specific binding described antigen The selection of one or more antibody in storehouse, and the success of immunne response is to be at least partially based on described antibody specificity identification Antigen that (and finally eliminate) stimulates also ignores the ability of other molecules in described antibody environment.

The serviceability of the antibody of specific recognition a kind of particular target antigen result in exploitation monoclonal antibody technique.Existing , the hybridoma technology of standard can make have monospecific antibody for purpose antigen and be produced.Recently, recombinant antibodies The most external antibody library screening of technology was developed already.These technology can make have single specificity for purpose antigen equally Antibody produced.

In the method for the invention, can allow purpose antigen in vivo or interaction in vitro is in antibody library.

According to an embodiment, by allowing described antigenic action in antibody library with antigen immunity inoculation animal in vivo. Set up many hybridomas by the lymphocyte of animal additionally, this vivo approaches can comprise and select secretion and described antigenic specificity In conjunction with the specific cross tumor of antibody.Treat the animal of immunity inoculation it may be that such as mice, rat, rabbit, chicken, camel or sheep, Can be maybe the transgenic variant of any above-mentioned animal mentioned, the transgenic such as with human immunoglobulin gene be little Mus, it produces people's antibody after antigenic stimulus.Other kinds of can be included employment periphery monokaryon blood by the animal of immunity inoculation Cell (chimeric hu-PBMC SCID mice) or lymphoid cell or its front volume reconstruction suffer from Reconstruction in Sever Combined Immunodeciency (SCID) mice, and process with lethal total irradiation, then be used to from suffering from Reconstruction in Sever Combined Immunodeciency (SCID) The medullary cell of mice be protected from the mice (" Trimera " system) of radiation portability function human lymphocyte subsequently. The another type of animal treating immunity inoculation is that the endogenous gene encoding purpose antigen in its genome turns off (clpp gene Remove) animal (such as mice), such as pass through homologous recombination so that with antigen immune inoculate after, described animal is by described antigen It is identified as xenogenesis.It will be apparent to one skilled in the art that by utilizing known screening technique to include but not limited to ELISA and dot blot can identify and select the polyclone through the method generation or monoclonal antibody.

According to another embodiment, by with antigen selection recombinant antibodies library so that described antigen acts in vitro In antibody library.Recombinant antibodies library can be on such as phage surface or in yeast cell surface or at bacterial cell surface Upper expression.In multiple embodiments, recombinant antibodies library is such as scFv library or Fab library.According to another embodiment party Case, antibody library is expressed as RNA-protein fusions.

Internal and in vitro method combination is included for producing the another kind of method of the antibody of the present invention.Such as, can pass through With antigen vivo immunization inoculation animal and then prepare lymphoid cell from described animal with the screening in vitro of described antigen Recombinant antibodies library or single domain antibodies library (as containing heavy chain and/or light chain), make described antigen be acted on antibody library.Root According to another kind of method, by with antigen vivo immunization inoculation animal and then allow the weight of the lymphoid cell being produced from described animal Affinity maturation is stood in group antibody library or Dan Qu library, makes described antigen be acted on antibody library.According to another kind of method, By inoculating animal by antigen vivo immunization, then select the individual antibody produced cell of secretion purpose antibody and from described selection Cell cDNA s obtain heavy chain and variable region of light chain (as by utilizing PCR), and table in mammalian host cell in vitro Reach the variable region (this is referred to as being chosen for lymphocyte antibody method or SLAM) of described heavy chain and light chain, thus can select further And operation is chosen for antibody gene sequences, described antigen is made to be acted on antibody library.Additionally, can be through at mammalian cell Middle expression encoding heavy chain and the antibody gene of light chain also select sucklings of antibody that those secretions have expectation binding affinity to move Thing cell, selects monoclonal antibody by expression cloning.

Present invention provide for screening and the regulation antigen of anti-screening.Therefore, those are likely selected according to the present invention Polyclone and the monoclonal antibody of A β (20-42) ball aggressiveness is combined with binding affinity as defined above.

Can be used for producing polytype antibody for producing the method for the present invention of antibody.These include monoclonal, Particularly recombinant antibodies, the most especially people's antibody, chimeric antibody, humanized antibody and CDR grafted antibody, and they Antigen-binding portion thereof.

The invention further relates to produce the hybridoma of the monoclonal antibody of (secretion) present invention.The hybridoma of the present invention Including those be selected from PTA-7241, PTA-7239, PTA-7240, PTA-7242, PTA-7408, PTA-7409, PTA-7405, The hybridoma specified by ATCC deposit number of PTA-7809, PTA-7810 and PTA-7851.

Notice that the antibody of the present invention also can react with the A beta form in addition to A β ball aggressiveness described herein (i.e. to tie Close).These antigens can be or can not be oligomerize or ball is multimerizing.Therefore, the antigen of the antibodies of the present invention A beta form including the ball mer epitopes of any antibody response comprised with the present invention.Above-mentioned A beta form include truncate and non-section Short A β (X-Y) form (with X and Y be as defined above), such as A β (20-42), A β (20-40), A β (12- 42), A β (12-40), A β (1-42) and A β (1-40) form, premise is that described form comprises ball mer epitopes.

The invention still further relates to the compositions of a kind of antibody comprising the present invention as defined above or its antigen-binding portion thereof.

According to a specific embodiment, described compositions is a kind of antibody comprising the present invention or antigen-binding portion thereof And the pharmaceutical composition of pharmaceutically acceptable carrier.

Antibody or the antigen-binding portion thereof of the present invention preferably can neutralize it in vitro and in vivo and be combined as defined above The activity of A β ball aggressiveness or derivatives thereof.Therefore, described antibody or antigen-binding portion thereof can be used for suppressing described ball aggressiveness or The activity of its derivant, such as in the goods containing described ball aggressiveness or derivatives thereof the most described ball aggressiveness or its spread out In the biological individual human existed or other mammals.

According to an embodiment, the present invention relates to a kind of active method suppressing described ball aggressiveness or derivatives thereof, The method includes allowing the antibody of the present invention or its antigen-binding portion thereof act on ball aggressiveness or derivatives thereof, so that suppressing described ball The activity of aggressiveness or derivatives thereof.The most described activity can be suppressed in vitro.Such as, the antibody of the present invention or antigen can be tied Conjunction part is added to containing or suspects that the goods containing described ball aggressiveness or derivatives thereof such as derive from individuality or cell is cultivated In the sample of thing, in order to suppress the activity of ball aggressiveness or derivatives thereof described in described sample.Alternatively, ball aggressiveness or its derive The activity of thing can be internal suppressed at individuality.

Therefore, the invention further relates to antibody as defined above or antigen-binding portion thereof in preparation for treatment or pre- Anti-amyloidosis, is especially selected from the amyloidosis of the amyloidosis of Alzheimer and mongolism Pharmaceutical composition in application.Therefore, an aspect of application of the present invention is a kind for the treatment of or prevention this treatment of needs Or the side of the amyloidosis of amyloidosis, particularly Alzheimer or mongolism in the individuality of prevention Method, it includes giving individual antibody or antigen-binding portion thereof as defined above.Use described antibody or antigen-binding portion demultiplexing In treatment and the amyloidosis of especially prophylaxis of amyloidosis disorders, particularly Alzheimer or mongolism Disease, especially for passive immunization.Therefore, treatment or prophylaxis of amyloidosis disorders, particularly Alzheimer or In the method for the amyloidosis of mongolism, in needing the individuality of this treatment or prevention, give this individuality antibody or anti- One purpose passive immunity of former bound fraction is individual comprehensive to resist amyloidosis, particularly Alzheimer or Tang Shi The amyloidosis of simulator sickness.

Antibody or the antigen-binding portion thereof of the present invention preferably can detect it in vitro and in vivo and be combined as defined above A β ball aggressiveness or derivatives thereof.Therefore, described antibody or antigen-binding portion thereof can be used for detecting described ball aggressiveness or it derives Thing, such as in the goods containing described ball aggressiveness or derivatives thereof or the most described ball aggressiveness or derivatives thereof exist people In individuality or other mammals.

According to an embodiment, the present invention relates to a kind of method detecting described ball aggressiveness or derivatives thereof, the method Act on ball aggressiveness or derivatives thereof including allowing the antibody of the present invention or its antigen-binding portion thereof so that with described ball aggressiveness or its Derivant combines (and being therefore preferably formed as comprising antibody or its antigen-binding portion thereof and the complex of ball aggressiveness or derivatives thereof). Described ball aggressiveness can detect the most in vitro.Such as, the antibody of the present invention or antigen-binding portion thereof can be added to goods, such as Containing or under a cloud containing described ball aggressiveness or derivatives thereof from individual sample or cell culture, in order to detection is described Described ball aggressiveness or derivatives thereof in goods.Alternatively, can be at individual vivo detection ball aggressiveness or derivatives thereof.

Therefore, the invention further relates to antibody as defined above or antigen-binding portion thereof in preparation for diagnosing starch Application in the compositions of the amyloidosis of sample degenerative disease, particularly Alzheimer or mongolism.The present invention One aspect of described application is that a kind of diagnosis suspection suffers from amyloidosis, and particularly Alzheimer or Tang Shi is comprehensive The amyloid of amyloidosis in the individuality of the amyloidosis levied, particularly Alzheimer or mongolism The method of degenerative disease, it includes that giving individual antibody or antigen-binding portion thereof detection as defined above comprises antibody or antigen , there is this complex and then indicate in individuality and there is amyloidosis, particularly in the formation of the complex of bound fraction and antigen Alzheimer or the amyloidosis of mongolism.Another aspect of application of the present invention is a kind of diagnosis Suspect suffer from amyloidosis, particularly starch in the individuality of the amyloidosis of Alzheimer or mongolism The method of the amyloidosis of sample degenerative disease, particularly Alzheimer or mongolism, it includes providing from individual The sample of body, by this sample and antibody as defined above or antigen binding portion thereof and detect and comprise antibody or antigen is combined , there is this complex and then indicate in individuality and there is amyloidosis in the formation of part and the complex of antigen, particularly A Er Ci Haimo disease or the amyloidosis of mongolism.

Detailed Description Of The Invention

In vitroimmunoassay the method such as ELISA, Dot blot of available standard or resisting of the BIAcore assay present invention The binding affinity (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) of body.About Further description, seesU., (1993) Ann.Biol.Clin.51:19-26 is waited；U., etc. (1991) Biotechniques 11:620-627；Johnsson, B., wait (1995) J.Mol.Recognit.8:125-131 and Johnsson, B., wait (1991) Anal.Biochem.198:268-277.

According to a specific embodiment, affinity defined herein refers to by carrying out as described in Example 8 Dot blot and evaluate its value obtained by photodensitometry.A particular according to the present invention, passes through Dot blot measures binding affinity and includes the following: a certain amount of antigen (such as, A β (X-Y) ball aggressiveness as above, A β (X-Y) monomer or A β (X-Y) fibril) or easily, such as at 20mM NaH₂PO₄, 140mM NaCl, pH 7.4,0.2mg/ Ml BSA is diluted to such as 100pmol/ μ l, 10pmol/ μ l, 1pmol/ μ l, 0.1pmol/ μ l and the antigen of 0.01pmol/ μ l Its suitable dilution print nitrocellulose membrane of concentration is upper, then with milk closing membrane to stop non-specific binding and to wash Washing, then contacted with purpose antibody by film, then second antibody and chrominance response by using conjugated enzyme detect the latter；On rule Under fixed antibody concentration, the amount of antibodies provides affinity and measures.Therefore, two kinds of different antibody are for a kind of target or Under the conditions of kind of antibody is defined herein as the most identical Dot blot for the relative affinity of two kinds of different targets, Two kinds of antibody-target is used to combine the ratio of the amount of antibody that viewed respective target combines.Unlike class based on Western blotting Like method, dot blotting by measure antibody to exist with native conformation to the affinity of targeting；Unlike ELISA method, speckle Blotting is as broad as long on the affinity between different targets and substrate, thus can carry out more accurately between different targets Relatively.

As used herein, term " K_d" mean specific antibodies-AI as known in the art Dissociation constant.

The antibody that the antibody of the present invention preferably separates." antibody of separation " means have binding affinity as above And it is essentially free of the antibody of other antibody with different binding affinity.Term " is essentially free of " and herein means it In at least 95% the antibody of antibody, the antibody of preferably at least 98% and more preferably at least 99% to have desired combination affine The antibody preparation of power.Additionally, the antibody separated can substantially not contain other cellular materials and/or chemicals.

The antibody of the separation of the present invention includes monoclonal antibody.As used herein, with containing different aminoacids sequence " polyclone " antibody preparation of the mixture of the antibody of row differs widely, and it is identical that " monoclonal antibody " means that antibody is enjoyed The antibody molecule goods of heavy chain and identical light-chain amino acid sequence.Can pass through some new techniques such as phage, antibacterial, yeast or Ribosomal display and by the antibody originated by hybridoma illustratively (as by hybridoma technology such as standard Resisting secreted by hybridoma prepared by Kohler and Milstein hybridoma method ((1975) Nature 256:495-497) Body) traditional method produce monoclonal antibody.Therefore, the antibody in the non-hybridoma source with identical sequence is the most single at this Clonal antibody, although it can be obtained by non-classical methodology, term " monoclonal " is not limited to the antibody in hybridoma source, But for referring to all antibody deriving from a nucleic acid clone.

Therefore, the monoclonal antibody of the present invention includes recombinant antibodies.Here, term " is recombinated " refers to such as pass through chemosynthesis Or operate any of the different sequence sections being separated of two obtained by the nucleic acid segment separated by technique for gene engineering Artificial combination.Particularly, term " recombinant antibodies " refers to the antibody being produced by recombinant means, express, generate or being separated, such as, make The antibody expressed with the recombinant expression carrier being transfected in host cell；It is isolatable from the antibody of restructuring combinatorial antibody library；Separate (see for example Taylor, L.D. from the antibody of the transgenic animal (such as mice) proceeding to human immunoglobulin gene, wait (1992) Nucl.Acids Res.20:6287-6295)；Or with wherein by specific Ig gene sequences (such as people's immunity ball Protein gene sequence) any other method of carrying out assembling with other DNA sequence antibody of producing, express, generate or separating.Weight Group antibody includes such as chimeric antibody, CDR grafted antibody and humanized antibody.Those skilled in the art are it should be recognized that at Heterologous System The middle derivative monoclonal antibody of conventional hybridoma of expressing needs to produce recombinant antibodies, though the amino of obtained antibody protein Acid sequence does not change or is intended to change.

In a specific embodiment of the invention, antibody is humanized antibody.

According to multiple embodiments, antibody can comprise the aminoacid sequence being entirely derived from single species, such as people's antibody or Mouse antibodies.According to other embodiments, antibody can be chimeric antibody or CDR grafted antibody or different types of humanization resists Body.

Term " antibody " means that the immunoglobulin being made up of 4 polypeptide chains (two weight (H) chain and two light (L) chains) divides Son.Chain is generally connected to each other by disulfide bond.Every heavy chain by described heavy chain variable region (being referred to as HCVR or VH at this) and The constant region composition of described heavy chain.CH is made up of three districts CH1, CH2 and CH3.Every light chain is by described light chain The constant region composition of variable region (being referred to as LCVR or VL at this) and described light chain.Constant region of light chain is made up of CL district.VH and VL District can be further separated into the hypervariable region of referred to as complementary determining region (CDRs) and the alternatively distributed conserved region of referred to as framework region (FR). Therefore, each VH and VL district is made up of three CDR and four FR of arrangement from N-terminal to C-terminal in the following order: FR1, CDR1、FR2、CDR2、FR3、CDR3、FR4.This structure is well-known for those skilled in the art.

" antigen-binding portion thereof " (or being referred to as " antibody moiety ") of term antibody refers to the antibody of one or more present invention Fragment, described fragment still has binding affinity as defined above.The fragment of complete antibody has shown can realize antibody Antigen combined function.According to " antigen-binding portion thereof " of term antibody, the example of binding fragment includes (i) Fab fragment, i.e. by The monovalent fragment of VL, VH, CL and CH1 district composition；(ii)F(ab’)₂Fragment, i.e. comprises two by disulfide bridge bond in hinge region The bivalent fragment of the Fab fragment being connected to each other；(iii) the Fd fragment being made up of VH and CH1 district；(iv) by the FL of antibody single armed and The Fv fragment of VH district composition；(v) by VH district or VH, CH1, CH2, DH3, or VH, CH2, CH3 composition dAb fragment (Ward etc., (1989) Nature 341:544-546)；And the complementary determining region (CDR) that (vi) separates.Although the Liang Ge district of Fv fragment is (i.e. VL and VH) it is single coded by said gene, but use the joint such as many-G of synthesis₄S aminoacid sequence and recombination method can be by They link together further so that they can combine as wherein VL and VH district to form monovalent molecule and (being referred to as ScFv (ScFv)；See for example (1988) the Science 242:423-426 such as Bird；With (1988) such as Huston Proc.Natl.Acad.Sci.USA 85:5879-5883) wall scroll protein chain and prepared." the antigen knot of term antibody Close part " it is also intended to include such single-chain antibody.The single-chain antibody of other forms such as " double antibody " is included in this similarly. Double antibody is bivalence, bi-specific antibody, and wherein VH and VL district expresses on single polypeptide chain, but can be combined in for the two The joint peptide used for district on same chain is the shortest, thus forces described district to match from the complementary region of different chains and formed Two antigen-binding sites (see for example Holliger, P., wait (1993) Proc.Natl.Acad.Sci.USA 90:6444- 6448；Poljak, R.J., wait (1994) Structure 2:1121-1123).Constant region for immunoglobulin refers to heavy chain or light chain Constant region.Human IgG heavy chain and chain constant region amino acid sequence are known in the art and are shown in Table 1.

Table 1: human IgG CH and constant light chain sequences

Additionally, the antibody of the present invention or its antigen-binding portion thereof can be by described antibody or antibody moiety and one or many Kind of other albumen or peptide covalently or non-covalently associate the part of the bigger immunoadhesin molecule formed.With above-mentioned immunity What adhesin molecule was relevant is uses streptavidin core region so that preparation four poly-scFv molecules (Kipriyanov, S.M., (1995) Human Antibodies and Hybridomas 6:93-101 is waited) and use cysteine residues, mark Note peptide and C-terminal polyhistidine base such as six histidine disjunction mark label are to produce bivalence and biotinylated scFv molecule (Kipriyanov, S.M. wait (1994) Mol.Immunol.31:1047-1058).

Term " people's antibody " refers to as (seen Kabat by Kabat etc., wait (1991) Sequences of Proteins Of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No 91-3242) described by, its variable region and constant region corresponding to or derive from human germline immunoglobulin The antibody of sequence.But, people's antibody of the present invention can contain such as in CDR, and is not particularly people's germline in CDR3 Amino acid residue coded by immunoglobulin sequences is (the most by external random or site-specific mutagenesis or internal somatic cell The sudden change that sudden change is imported).The recombinant human antibody of the present invention has variable region, and also can comprise from human germline immunoglobulin The constant region of sequence (sees Kabat, E.A., waits (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No 91- 3242).But, according to specific embodiment, above-mentioned recombinant human antibody is carried out in vitro mutagenesis (if or use proceed to people Ig The animal of sequence gene, then carry out internal somatic mutagenesis) so that although the aminoacid sequence in recombinant antibodies VH and VL district be with People's germline VH is relevant with VL sequence or derives from it, but is not naturally occurring in the sequence in internal people's antibody germline storehouse.According to Specific embodiment, such recombinant antibodies is to select mutation or back mutation or both results.Preferably, mutation Cause compared with parental antibody the affinity for target bigger, and/or the affinity for non-target structure is less.

Term " chimeric antibody " refers to containing from the heavy chain of a kind of species and light-chain variable sequence and from another thing The antibody of the constant-region sequences planted, such as, have the Mus heavy chain and the antibody of variable region of light chain being connected with human constant region.

Term " CDR grafted antibody " refers to comprise the heavy chain from a kind of species and light-chain variable sequence, but wherein VH and/ Or the substituted antibody of CDR sequence that one or more CDR region sequences of VL are another species, such as have one of them or many Individual Mus CDR (such as CDR3) is for people's CDR sequence substituted Mus heavy chain and the antibody of variable region of light chain.

Term " humanized antibody " refers to containing from non-human species (such as mice, rat, rabbit, chicken, camel, sheep or goat) Heavy chain and light-chain variable sequence, but at least a part of which a part VH and/or VL sequence be altered to more " proper manners ", I.e. it is more closely similar to the antibody of human germline variable region sequence.A type of humanized antibody is the most people's CDR sequence to have been inserted Inhuman VH and VL sequence replaces the CDR grafted antibody of corresponding non-human CDR sequences.

In this article, " Kabat numbering, " Kabat definition " and " Kabat labelling " are used interchangeably term.These are ability Term recognized by territory refers to that numbering is residual with other aminoacid in antibody or the heavy chain of its antigen-binding portion thereof and variable region of light chain Base compare the amino acid residue of more variable (i.e. hypermutation) system (Kabat etc. (1971) Ann.NY Acad, Sci.190: 382-391 and Kabat, E.A., wait (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S.Department of Health and Human Services, NIH Publication No 91-3242).For Variable region of heavy chain, hypervariable region CDR1 from the 31st to the 35th, aminoacid, CDR2 from the 50th to the 65th, aminoacid, CDR3 from The 95th to the 102nd, aminoacid.For variable region of light chain, hypervariable region CDR1 from the 24th to the 34th, aminoacid, CDR2 from The 50th to the 56th, aminoacid, CDR3 is from the 89th to the 97th, aminoacid.

As used herein, term " receptor " and " receptor antibody " refer to provide or coding at least 80%, at least 85%, The antibody of the aminoacid sequence of one or more framework regions of at least 90%, at least 95%, at least 98% or 100% or nucleic acid sequence Row.In certain embodiments, term " receptor " refers to offer or the antibody amino acid of encoding constant regions or nucleotide sequence.Another In individual embodiment, term " receptor " refers to provide or encode one or more framework region and the antibody amino acid of constant region or nucleic acid Sequence.In a specific embodiment, term " receptor " refer to provide or coding at least 80%, preferably at least 85%, at least 90%, people's antibody amino acid of the aminoacid sequence of one or more framework regions of at least 95%, at least 98% or 100% or core Acid sequence.According to this embodiment, receptor can be containing at least 1, at least 2, at least 3, at least 4, at least 5 or at least Amino acid residue on 10 one or more ad-hoc locations being not present in people's antibody.Acceptor framework region and/or receptor are constant District can be such as to derive from or be derived from germline antibody gene, ripe antibody gene, functional antibodies (such as this area crowd institute Antibody in known antibody, exploitation or commercial antibody).

As used herein, the complementary determining region in term " CDR " refers to antibody variable sequence.At each heavy chain with light There are three CDR in chain variable region, for each variable region, it is referred to as CDR1, CDR2 and CDR3.As used herein, art Language " CDR group " refers to one group of three CDR being present in the single variable region of energy conjugated antigen.According to different systems to these CDR boundary really has made different definition.This system (Kabat etc., Sequences of Proteins that Kabat describes Of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provide not only and be applicable to the clear and definite residue numbering system of any antibody variable region, additionally provide restriction this three The accurate residue border of individual CDR.These CDR can be described as Kabat CDR.Chothia and colleague (Chothia&Lesk, J.Mol.Biol.196:901-917 (1987) and Chothia etc., Nature 342:877-883 (1989)) find Kabat Although a little position of certain in CDR differs greatly on amino acid sequence level, but have employed almost identical peptide Conformation of the main chain. This little position is named as L1, L2 and L3 or H1, H2 and H3, and wherein " L " and " H " refers to light chain and heavy chain district respectively.These districts Can be described as Chothia CDR, it has the border overlapping with Kabat CDR.Other boundary definitions overlapping with Kabat CDR CDRs has been Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262 (5): 732-45 (1996)) described.While it is true, other CDR boundary definitions can strictly in accordance with any one of said system, But still overlapping with Kabat CDR, although the most not clear according to prediction or specific residue or residue group or the most whole CDR Development ring antigen combine experiment conclusion they can be shorter or longer.Available according to any one by method in this article CDR defined in these systems, although preferred embodiment make use of the CDR that Kabat or Chothia defines.

As used herein, term " specification " residue refer to define as Chothia etc. (J.Mol.Biol.196: 901-907(1987)；Chothia etc., J.Mol.Biol.227:799 (1992), be both herein incorporated by reference) determined Residue in the CDR of the specific Canonical CDR structure of justice or in framework region.According to Chothia etc., the key of many antibody CDR Although position differs greatly on amino acid sequence level, but there is almost identical peptide Conformation of the main chain.Each norm structure is advised The peptide main chain having determined the mainly continuous section of one group of looped amino acid residue of shape reverses.

As used herein, term " donor " and " donor antibody " refer to provide the antibody of one or more CDR.One In individual preferred embodiment, donor antibody is a kind of from the species being different from the antibody that framework region is derived from or derives from Antibody.In the context of humanized antibody, term " donor antibody " refers to provide the non-human antibody of one or more CDR.

As used herein, term " framework region " or " framework region sequence " refer to that variable region deducts the sequence remained by CDR Row.Because different system can be used to determine the definite definition of CDR sequence, therefore the implication of framework region sequence can correspondingly be carried out not Same explanation.6 CDRs (CDR-L1 ,-L2 and the-L3 of light chain and CDR-H1 ,-H2 and the-H3 of heavy chain) are also by light chain and heavy chain Framework region on every chain is divided into four subprovinces (FR1, FR2, FR3 and FR4), and wherein CDR1 is between FR1 and FR2, CDR2 Between FR2 and FR3, and CDR3 is between FR3 and FR4.Be not specified by specific subprovince be FR1, FR2, FR3 or FR4, other people mentioned framework region represents the FR ' s combined in the single variable region of naturally occurring immunoglobulin chain,. As used herein, FR represents in four subprovinces, and FR represents two in four subprovinces constituting framework region Or it is more.

People's heavy chain and light chain acceptor sequence are known in the art.

As used herein, term " germline antibody gene " or " genetic fragment " refer to as not having experience to cause for spy Determine the immunoglobulin sequence coded by non-lymphoid cell of the gene rearrangement of immunoglobulin expression and the maturation process of sudden change Row (see such as Shapiro etc., Crit.Rev.Immunol.22 (3): 183-200 (2002)；Marchalonis etc., Adv Exp Med Biol.484:13-30 (2001)).The advantage that the multiple embodiment of the present invention is provided comes from such discovery, i.e. Compared with ripe antibody gene, it is special that germline antibody gene more likely preserves individual basic amino acid sequence structure in species Levy, therefore be less likely to be identified as non-self antibody when in these species.

As used herein, term " crucial " residue refers to antagonist, particularly the binding specificity of humanized antibody And/or affinity has some residue in the variable region of bigger impact.Key residues include but not limited to following one or Multiple: the residue adjacent with CDR, possible glycosylation site (it can be N-or O-glycosylation site), rare residue, can be with Between residue, canonical residue, variable region of heavy chain and variable region of light chain that the residue of AI, energy and CDR interact Residue in contact residues, vernier (Vernier) district and defining at variable region of heavy chain CDR1 Yu Kabat of Chothia definition The first heavy chain framework region between residue in overlapping district.

As used herein, term " humanized antibody " particularly relates to a kind of specific binding with purpose antigen immune And the framework region (FR) that comprises the aminoacid sequence substantially with people's antibody and the aminoacid sequence substantially with non-human antibody The antibody of the complementary determining region (CDR) of row or its variant, derivant, analog or fragment.As used herein, at CDR In context, term " substantially " refers to that CDR has at least 80%, preferably at least 85%, at least 90%, at least 95%, at least The aminoacid sequence of the 98% or at least 99% same aminoacid sequence in non-human antibody CDR.Humanized antibody comprises substantially All of at least one, and particularly two variable regions (Fab, Fab ', F (ab ') 2, FabC, Fv), the most all or basic Upper all of CDR region corresponds to those CDR region of non-human immunoglobulin (i.e. donor antibody) and all of or substantially All of framework region is all those framework region consensus sequences of human normal immunoglobulin.Preferably, humanized antibody also comprises at least A part constant region for immunoglobulin (Fc), the usually constant region of human normal immunoglobulin.In certain embodiments, people Source antibody contains the variable region of light chain and at least heavy chain.Antibody may also include the CH1 of heavy chain, hinge, CH2, CH3 and CH4 District.In certain embodiments, humanized antibody contains only humanized light chain.In certain embodiments, humanized antibody Contain only humanized heavy chain.In certain embodiments, humanized antibody contain only humanized variable region of light chain and/or Humanized heavy chain.

Humanized antibody is selected from any kind of immunoglobulin, including IgM, IgG, IgD, IgA and IgE, Yi Jiren The immunoglobulin of what subclass, includes but not limited to IgG 1, IgG2, IgG3 and IgG4.

The framework region of humanized antibody and CDR region need not accurately corresponding to parental array, such as donor antibody CDR or total Framework region can be by replacing, inserting and/or lack at least one amino acid residue and obtain mutation so that CDR in this site or Framework region residue is not the most corresponding to donor antibody or total framework region.But, in a preferred embodiment, above-mentioned prominent Become and be not intended to be widely.In general, at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least The humanized antibody residue of 95% should be corresponding to those residues of parent FR and CDR sequence.As used herein, term " total framework region " refers to the framework region existed with total immunoglobulin sequences.As used herein, term " total immunity Globin sequence " refer to the sequence that formed by modal aminoacid (or nucleotide) in the immunoglobulin sequences family being correlated with (see such as Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987).In immunoglobulin class, each position in consensus sequence is modal in this position in this family Occupied by aminoacid.In the case of two aminoacid occupy with same frequency, all may be included in consensus sequence.

As used herein, " vernier (Vernier) " district refer to as Foote and Winter (1992, J.Mol.Biol.224:487-499, it is herein incorporated by reference) described in the scalable CDR structure of a subgroup and adjust The whole framework region residue making to be suitable to antigen.Vernier (Vernier) district residue constitutes the floor of a supporting CDR and can affect CDR structure And the affinity of antibody.

Term " epi-position " includes any polypeptide determinant being combined with specific for immunoglobulin.In some embodiment In, Epitopic determinants includes the chemically reactive surface group such as aminoacid of molecule, sugar side chain, phosphoryl or sulfonyl, and In certain embodiments, can have specific three dimensional structure characteristic and/or specific charging characteristic.Epi-position is to be tied by antibody One region of the antigen closed.In certain embodiments, when antibody in the complex mixture of albumen and/or macromole preferential When identifying its target antigen, just say this antibody specificity conjugated antigen.

When referred to herein, term " polynucleotide " means two or more nucleotide (ribonucleotide or de- The modified forms of the nucleotide of oxygen nucleotide or any type) Multimeric forms.This term includes strand and double chain form DNA but preferably double-stranded DNA.

As used herein, term " polynucleotide of separation " should mean according to its polynucleotide originated (as Genome, cDNA or synthesis source, or any combinations thereof), " polynucleotide of separation " and these " many nucleoside separated Acid " existing for nature in all or part of of simultaneous polynucleotide unrelated；Optionally with it in nature Unrelated polynucleotide are correlated with；Or the part not as bigger sequence is present in nature.

As used herein, term " carrier " means a kind of nucleic acid that can transport its nucleic acid already connected another kind of Molecule.A type of carrier is " plasmid ", its circular double stranded DNA referring to wherein to connect extra region of DNA section.Another kind The carrier of type is viral vector, and the most extra region of DNA section may be connected in viral genome.Some carrier can import at them Host cell in independently replicate (such as there is bacteria carrier and the episomal mammalian vectors of bacterial origin of replication).Other Carrier (such as non-add type mammalian vector) is upon importing in the genome that just may be incorporated into host cell in host cell, thus Replicate together with host genome.Additionally, some carrier can instruct the expression of its gene being operatively connected.Above-mentioned carrier is at this Referred to herein as " recombinant expression carrier " (or being referred to as " expression vector ").It is said that in general, expression vector leads in recombinant DNA technology It is often to apply with plasmid form.In the description of the present invention, " plasmid " and " carrier " uses convertibly, because plasmid is carrier Most-often used form.But, the invention is intended to include that the expression vector such as viral vector of other forms above-mentioned (lacks as replicated Swaged retrovirus, adenovirus and adeno associated virus), it provides identical effect.

Term " is operably connected " and refers to that a kind of component described in it is to allow that they rise in mode expected from them The neighbouring relationship that the relation of effect exists.Controlling the sequence coded sequence that " is operably connected " is the most i.e. in phase It is dissolved in what the expression controlling to realize coded sequence under conditions of sequence connected.The sequence that " is operably connected " includes adjacent mesh The expression control sequenc of gene and trans-acting or control the expression control sequenc of genes of interest a long way off.Such as institute herein Using, term " expression control sequenc " refers to that the coded sequence connected by affecting them is expressed and the necessary many nucleoside of processing Acid sequence.Expression control sequenc includes suitable transcriptional start point, terminal, promoter and enhancer sequence；Effective RNA processing letter Number such as montage and polyadenylation signal；Stablize the sequence of Cytoplasm mRNA；(i.e. Kozak has the sequence of enhancing translation efficiency Sequence)；Improve the sequence of protein stability；And when needed, increase the sequence of protein excretion.Depend on host organisms State the characteristic difference controlling sequence；In prokaryote, above-mentioned control sequence generally includes promoter, ribosome binding site Point and transcription terminator；In eukaryote, above-mentioned control sequence generally includes promoter and transcription terminator.Term " control sequence " is intended to include that it exists expressing and process requisite element, and to may also include its existence be favourable volume External component, such as targeting sequencing and fusion partner sequence.

As defined herein, the process referring to that any foreign DNA enters host cell " is converted ".Use this area crowd institute Known multiple method can convert under natural or artificial condition.Conversion can be dependent on any of for by external source core Acid sequence inserts the method in protokaryon or eukaryotic host cell.The method is to carry out selecting based on host cell to be converted And may include but be not limited to virus infection, electroporation, lipofection and particle bombardment.Above-mentioned " conversion " cell includes wherein The DNA inserted can be as autonomously replicating plasmid or the cell of the stable conversion replicated as a host chromosome part.They are also Cell including DNA or RNA that the most limited a period of time transient expression inserts.

As used herein, term " recombinant host cell " (or being referred to as " host cell ") means that foreign DNA is Import cell therein.Should be understood that above-mentioned term not only means specific subject cell, after also referring to such cell Generation.Owing to sudden change or some modification of environmental effect can occur in offspring, the most above-mentioned offspring can be differently configured from parent Cell, but be included in the scope of " host cell " as used herein, the term.Preferably, host cell includes being selected from The protokaryon of any life circle and eukaryotic cell.Preferably eukaryotic cell includes protista, fungus, plant and animal cell.? Preferably host cell includes but not limited to prokaryotic cell system escherichia coli；Mammal cell line CHO, HEK 293 and COS； Insect cell line Sf9 and fungal cell's saccharomyces cerevisiae (Saccharomvces cerevisiae).

Standard technique can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and converts (as electroporation, lipid turn Dye).Can be according to manufacturer's operation instructions or as generally carried out in this area or to carry out enzymatic as described in this article anti- Should and purification technique.Preceding technology and method can be generally according to conventional methods well-known in the art and as various conventional And description of the invention in full in institute quote as proof and discuss referring more particularly to performing described by document.See as Sambrook etc., Molecular Cloning:A Laboratory Manual (second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), it is incorporated at this as any purposes List of references.

As known in the art and used herein, " genetically modified organism " refers to have the cell containing transgenic Organism, wherein imports the transgene expression the most natural expression in this organism in organism (or ancestors of this organism) Polypeptide." transgenic " is a kind of DNA construction, and it is stable and is operationally integrated into the cell growing transgenic organism In genome, one or more cell types or tissue of transgenic organism instruct the expression of encoding gene product.

The method producing antibody of the present invention is described in down.At this, vivo approaches, in vitro method or combinations thereof are carried out Distinguish.

The method of some antibody producing the present invention is described in down.At this to vivo approaches, in vitro method or their group Conjunction makes a distinction.

Vivo approaches

Depending on the type expecting antibody, different host animals can be used for vivo immunization inoculation.Oneself expression can be used The host of the purpose antigen of endogenous form.Alternatively it is possible that the endogenous form of application target antigen is by the host of defect.Example As, the mice (i.e. knock out mice) of specific intrinsic protein defect has been made by homologous recombination at corresponding endogenous gene Show and can produce the humoral response for their albumen of immunity inoculation, and therefore can be used for producing the height for this albumen Affinity monoclonal antibody (see for example (1995) the J.Immunol.Methods 183:231-237 such as Roes, J.；Lunn, M.P. (2000) J.Neurochem.75:404-412 is waited).

In order to produce the non-human antibody of the present invention, multiple non-human mammal applies to the host that antibody produces.They Including mice, rat, chicken, camel, rabbit, sheep and goat (and their gene knockout model), although preferably mice is used for producing Raw hybridoma.Additionally, the nonhuman host animal expressing people's antibody library can be used for producing have bispecific for human antigen's People's antibody substantially.The non-human animal of the type includes the transgenic animal (such as mice) of carrier's immunoglobulin transgene (mosaic type hu-PBMC SCID mice) and people/mice irradiation chimera described more below.

According to an embodiment, it is inhuman suckling with the animal of A β (20-42) ball aggressiveness or derivatives thereof immunity inoculation Animal, preferably mice, it is transferred into human immunoglobulin gene and makes described non-human mammal manufacturer under antigenic stimulus Antibody.Typically, the immunoglobulin transgene of heavy chain and light chain that coding has people's germline configuration imports already to have changed and makes Obtain in the above-mentioned animal of its endogenous heavy chain and light chain gene seat inactivation.If stimulating above-mentioned animal with antigen (as with human antigen), Then produce the antibody (people's antibody) deriving from human normal immunoglobulin's sequence.By using the hybridoma technology of standard, it is possible to from The lymphocyte of above-mentioned animal prepares human monoclonal antibodies.About the transgenic mice with human normal immunoglobulin and at product Further describing of application in stranger's antibody see for example US 5,939,598, WO 96/33735, WO 96/34096, WO 98/24893 and WO 99/53049 (Abgenix Inc.), and US 5,545,806, US 5,569,825, US 5,625, 126, US 5,633,425, US 5,661,016, US 5,770,429, US 5,814,318, US 5,877,397 and WO 99/ 45962(Genpharm Inc.)；Referring further to MacQuitty, J.J. and Kay, R.M. (1992) Science 257:1188； Taylor, L.D. etc. (1992) Nucleic Acids Res.20:6287-6295；Lonberg, N. etc. (1994) Nature 368:856-859；Lonberg, N. and Huszar, D. (1995) Iht.Rev.Immunol.13:65-93；Harding, F.A. And Lonberg, N. (1995) Ann.N.Y.Acad.Sci.764:536-546；Fishwild, D.M. etc. (1996) Nature Biotechnology 14:845-851；Mendez, M.J. etc. (1997) Nature Genetics 15:146-156；Green, And Jakobovits, A. (1998) J.Exp.Med.188:483-495 L.L.；Green, L.L. (1999) J.Immunol.Methods 231:11-23；Yang, X.D. etc. (1999) J.Leukoc.Biol.66:401-410；Gallo, M.L. (2000) Eur.J.Immunol.30:534-540 is waited.

According to another embodiment, can be tool with the animal of A β (20-42) ball aggressiveness or derivatives thereof immunity inoculation There is the mice of Reconstruction in Sever Combined Immunodeciency (SCID), its employment periphery mononuclear blood cells or lymphoid cell or their precursor Rebuild.As certified, the people that the above-mentioned mice generation being referred to as mosaic type hu-PBMC SCID mice responds to antigenic stimulus exempts from Epidemic disease globulin.These mices and their further describing of application in producing antibody be see for example, Leader, K.A. (1992) Immunology 76:229-234 is waited；Bombil, F. etc. (1996) Immunobiol.195:360-375； Murphy, W.J. etc. (1996) Semin.Immunol.8:233-241；Herz, U. etc. (1997) Int.Arch.Allergy Immunol.113:150-152；Albeft, S.E. etc. (1997) J.Immunol.159:1393-1403；Nguyen, H. etc. (1997) Microbiol.Immunol.41:901-907；Arai, K. etc. (1998) J.Immunol.Methods 217:79- 85；Yoshinari, K. and Arai, K. (1998) Hybridoma 17:41-45；Hutchins, W.A. etc. (1999) Hybridoma 18:121-129；Murphy, W.J. etc. (1999) Clin.Immunol.90:22-27；Smithson, S.L. etc. (1999) Mol.Immunol.36:113-124；Chamat, S. etc. (1999) J.Infect.Diseases 180:268-277 and Heard, C. etc. (1999) Molec.Med.5:35-45.

According to another embodiment, with the animal of A β (20-42) ball aggressiveness or derivatives thereof immunity inoculation be one Process with the total irradiation of fatal dose, then make the medullary cell from Reconstruction in Sever Combined Immunodeciency (SCID) mice avoid spoke Penetrate and then carry out the mice transplanted with functional human lymphocyte.In order to by inoculating described mice also with purpose antigen immune The hybridoma technology then passing through use standard produces monoclonal antibody thus human monoclonal antibodies, employs referred to as Such chimera of Trimera system.For these mices and they are for producing application further of antibody Description see for example (1998) the Immunology 93:154-161 such as Eren, R.；Reisner, Y and Dagan, S. (1998) Trends Biotechnol.16:242-246；Ilan, E. etc. (1999) Hepatology 29:553-562；And Bocher, W.O. (1999) Immunology 96:634-641 is waited.

Initiateed by the antibody produced cell of internal generation, can by the technology utilizing standard be such as initially Kohler and Milstein (1975, Nature 256:495-497) is (referring also to (1981) J.Immunol 127:539-46 such as Brown； Brown etc. (1980) J Biol Chem 255:4980-83；Yeh etc. (1976) PNAS 76:2927-31；With (1982) such as Yeh Int.J.Cancer 29:269-75) described by hybridoma technology produce monoclonal antibody.Produce monoclonal antibody hybridoma Technology be fully known (generally to see R.H.Kenneth, in Monoclonal Antibodies:A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980)；E.A.Lerner (1981) Yale J.Biol.Med., 54:387-402；M.L.Gefter etc. (1977) Somatic Cell Genet., 3:231-36).In short, immortal cell line (usually myeloma) is gathered with the A β ball of the present invention The lymphocyte of the mammal of body or derivatives thereof immunity inoculation (usually splenocyte or lymph-node cell or peripheral blood lymph Cell) merge, and the culture supernatants of the hybridoma obtained by screening, in order to identify the monoclonal anti producing the present invention The hybridoma of body.Any one of many well-known methods for merging lymphocyte and immortal cell line are all Can be used for this purpose (referring also to (1977) Nature 266:550-52 such as G.Galfre；The Somatic Cell such as Gefter Genet., front quoting as proof；Lerner, Yale J.Biol.Med., front quote as proof；Kenneth, Hybridoma, in leading Card).Additionally, skilled artisan would appreciate that the different changes that there is said method, it is useful equally.Typical case Ground, immortal cell line (such as myeloma cell line) derives from the mammalian species identical with lymphocyte.Such as, can lead to Cross lymphocyte and the mouse cell lines of infinite multiplication of the mice of the immunogenic preparation immunity inoculation of the most personal present invention Merge and set up murine hybridoma.Preferably immortal cell line is to the culture medium containing hypoxanthine, aminopterin and thymidine The mouse myeloma cell line that (HAT culture medium) is sensitive.Any one of multiple myeloma cell line all can be given tacit consent to and is used as Fusion partner, such as P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma cell line.These myeloma Cell line is available from American type culture collection (ATCC), Rockville, MD.Typically, Polyethylene Glycol is used (PEG) murine myeloma cell sensitive for HAT-is merged with mouse boosting cell.Then HAT culture medium is utilized to select to be produced by fusion Raw hybridoma, thus kills the myeloma cell of fusion that is that do not merge and that do not go out product (after a few days due to not The splenocyte merged does not converts therefore dead).Produce by above-mentioned antibody screening hybridoma culture supemates is identified The hybridoma of the monoclonal antibody of the present invention, such as, surveyed by the Dot blot more than utilizing and described in embodiment 8 Fixed, in order to select those antibody with binding affinity as defined above.

Use above-mentioned vivo approaches created whole monoclonal antibody 5F7,10F11,7C6,4B7,6A2,2F2,4D10, 7E5,10C1 and 3B10 are also thus available from hybridoma as herein defined.

Similarly, further describing in detail as following, described hybridoma can be used as encoding light chain and/or heavy chain Nucleic acid source in case restructuring produce the present invention antibody.

In vitro method

As by immunity inoculation with select to produce the alternative of antibody of the present invention, can be by with A β (20-42) ball The combination immunoglobin libraries of aggressiveness or derivatives thereof screening restructuring, thus separates the immune ball with required binding affinity Protein library member, thus identify and separate the antibody of the present invention.For producing and screen the test kit of display libraries in market On can buy (such as Pharmacia recombinant phages antibody system, catalog number 27-9400-01；And StratagenePhage display test kit, catalog number 240612).In many embodiments, display libraries is scFv Library or Fab library.The most at large describe the display technique of bacteriophage for screening recombinant antibodies library.Can be particularly advantageously Example for generation and the method for screening antibodies display libraries and compound is found in the WO 92/ of such as McCafferty etc. 01047, US 5,969,108 and EP 589 877 (describe in detail scFv show), the US 5,223,409, US of Ladner etc. 5,403,484, US 5,571,698, US 5,837,500 and EP 436 597 (such as describing pIII to merge)；Dower's etc. WO 91/17271, US 5,427,908, US 5,580,717 and EP 527 839 (describing Fab in detail to show)；Winter etc. International publication WO 92/20791 and EP 368,684 (particularly describing the sequence of Cloning of Immunoglobulin variable region)； The US 5,885,793 and EP 589 877 of Griffiths etc. (describes in detail by using restructuring library Separated pin to resist people Former people's antibody)；The WO 92/09690 (particularly describing phage expression technology) of Garrard etc.；The WO of Knappik etc. 97/08320 (describing people's recombinant antibodies library HuCal)；The WO 97/29131 of Salfeld etc. (describes for human antigen The generation of the recombinant human antibody of (huamn tumor necrosis factory alpha) and the external affinity maturation of this recombinant antibodies) and Salfeld etc. U.S. Provisional Application No. 60/126,603 and patent application based on this (similarly describe for human antigen that (people is the thinnest Born of the same parents' interleukin-12) the generation of recombinant human antibody, also describe the external affinity maturation of this recombinant antibodies) in.

More descriptions in screening recombinant antibodies library are found in technical press, such as Fuchs etc. (1991) Bio/ Technology 9:1370-1372；Hay etc. (1992) Hum Antibod Hybridomas 3:81-85；Huse etc. (1989) Science 246:1275-1281；Griffiths etc. (1993) EMBO J 12:725-734；Hawkins etc. (1992) J Mol Biol 226:889-896；Clarkson etc. (1991) Nature 352:624-628；Gram etc. (1992) PNAS 89:3576- 3580；Garrard etc. (1991) Bio/Technology 9:1373-1377；Hoogenboom etc. (1991) Nuc Acid Res 19:4133-4137；Barbas etc. (1991) PNAS 88:7978-7982；The Nature such as McCafferty (1990) 348:552- In (2000) J.Mol.Biol.296:57-86 such as 554 and Knappik.

As the alternative of use phage display system, recombinant antibodies library can be at yeast cells or bacterial cell table Face is expressed.WO 99/36569 describes preparation the method screening the library expressed in yeast cell surface.WO 98/ 49286 describe the method preparing and screening the library expressed on bacterial cell surface in more detail.

In all of in vitro method, a kind of system of selection for being enriched with the recombinant antibodies with desired characteristic constitutes The major part of the method, it is commonly referred to " elutriation " often take to carry out being connected on the pillar of its substrate by target structure The form of affinity chromatograph.Then desired candidate molecules is individually measured they definitely and/or relative affinity, preferably as with Described in upper and embodiment 8 by utilizing the Dot blot of standard to measure.

Once identify and fully characterize the purpose antibody of combinatorial library, by utilizing the Protocols in Molecular Biology of standard to divide From encoding said antibody light chain and the DNA sequence of heavy chain, such as by displaying separated during utilizing comfortable library screening The PCR amplification of the DNA of packing material (such as phage).Can be used for preparing the gene of the encoding antibody light of PCR primer and heavy chain Nucleotide sequence is well known by persons skilled in the art.Multiple above-mentioned sequence description, in such as Kabat, E.A., waits (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of In Health and Human Services, NIH Publication No 91-3242 and ethnic group sequence library VBASE.

Can produce the present invention's by the gene of recombinant expressed encoding immune immunoglobulin light chains and heavy chain in host cell Antibody or antibody moiety.For recombinant expressed antibody, carry the light chain immunoglobulin of encoding said antibody with one or more With the recombinant expression carrier transfection host cell of the DNA fragmentation of heavy chain, in host cell, thus express light chain and heavy chain excellent They are secreted in the culture medium cultivating described host cell by choosing.Can be from this culture medium separation antibody.The restructuring of use standard Described gene, to obtain encoding antibody heavy and the gene of light chain, is inserted in recombinant expression carrier and by institute by DNA method State in vector introduction host cell.Such method is described in such as Sambrook, Fritsch and Maniatis (volume), Molecular Cloning；A Laboratory Manual, second edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F.M. etc. (volume) Current Protocols in Molecular Biology, Greene Publishing Associates, the US 4 of (1989) and Boss etc., in 816,397.

Once obtain the DNA fragmentation of VH and the VL section of coding purpose antibody, so that it may utilize the recombinant DNA technology of standard to enter DNA fragmentation described in single stepping, such as in order to be the gene of encoding full leng antibody chain, coding by the gene conversion of encoding variable regions The gene of Fab fragment or scFv gene.These operations include being operably connected VL-or VH-coding DNA fragment and encode another Plant the another kind of DNA fragmentation of albumen such as antibody constant region or flexible joint.Term " is operably connected " and is to be understood that at this For such implication, i.e. two DNA fragmentations are retained in frame so with the aminoacid sequence coded by described two DNA fragmentations A kind of mode connects.

By the another kind of DNA molecular in the VH-district of coding DNA with encoding heavy chain constant (CH1, CH2 and CH3) can be grasped Make ground to connect, the DNA in the coding VH district separated can be changed into the gene of encoding full leng heavy chain.The sequence of people's weight chain constant area gene Row are well-known (to see for example, Kabat, E.A., wait (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No 91-3242), and the DNA fragmentation in the described region of leap can be obtained by utilizing the PCR of standard to expand.Heavy chain Constant region can be from the constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD, preferably constant region from IgG, particularly IgG1 or IgG4.For obtaining the gene of encoding heavy chain Fab fragment, VH-coding DNA can be operably connected to On the another kind of DNA molecular of only encoding heavy chain constant CH1.

Can by VL-coding DNA is operably connected to encode constant region of light chain CL another kind of DNA molecular on, from And the DNA in the coding VL district separated is changed into the gene (and gene of coding Fab light chain) of encoding full leng light chain.People is light The gene order of chain constant region is well-known (to see Kabat, E.A., wait (1991) Sequences of Proteins Of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Pub. No 91-3242), and the DNA fragmentation crossing over described district can be by utilizing the PCR amplification acquisition of standard.Light chain is permanent Determining district can be κ or λ constant region, preferably κ constant region.

In order to produce scFv gene, can be operably connected to VH-and VL-coding DNA fragment encode flexible joint example Such as aminoacid sequence (Gly₄-Ser)₃Another fragment on so that VH and VL sequence is as having VL and VH district each other by described The continuous print single chain protein that flexible joint connects is expressed and (is seen (1988) the Science 242:423-426 such as Bird；Huston etc. (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883；McCafferty etc., Nature (1990) 348:552- 554)。

Can be had single district VH and VL of binding affinity as above from the separation of Dan Qu library by said method.Such as this Described in literary composition, there is expectation Liang TiaoVHDan district chain (with and without CH1) of binding affinity or two VL chains or one The pairing of VH chain and a VL chain can be used for the antibody of the present invention.

In order to express recombinant antibodies or the antibody moiety of the present invention, can be by coded portion or full-length light chains and the DNAs of heavy chain Insert in expression vector so that be operatively connected this gene and transcribe or translate control sequence to suitable.Within a context, term " it is operably connected " and should be understood to that such implication, i.e. antibody gene control sequence with the transcription and translation in carrier and fulfil The such a mode of its intended transcription and translation effect regulating described antibody gene connects in the carrier.

Preferably, expression vector and expression control sequenc is selected to make to be compatible to used expression host cell.Can be by The gene of encoding antibody light inserts in different carriers with the gene of encoding antibody heavy, or inserts same by the two gene In individual expression vector, this is common situation.(such as pass through by utilizing standard method to be inserted in expression vector by antibody gene Antibody gene segments and carrier connect the restricted cleavage site of complementation, or exists then without restricted cleavage site By connecting flush end).Expression vector can carry encoding antibody constant region before inserting the sequence of coding light chain and heavy chain the most Sequence.Such as, a kind of method is by VH and VL sequence is inserted the expression load being separately encoded heavy chain and constant region of light chain In body, connect VH section and CH section the most in the carrier and connect VL section and CL the most in the carrier Section, thus be full length antibody gene by VH and VL sequence transitions.

Additionally or alternatively, recombinant expression carrier codified is beneficial to the signal peptide that antibody chain is secreted from host cell.Can Described antibody chain encoding gene is cloned in carrier, thus by the N end of signal peptide and this antibody chain encoding gene to meet reading The form of frame connects.Signal peptide can be that immunoglobulin signal peptide or heterologous signal peptide are (i.e. from NIg albumen Signal peptide).In addition to antibody chain encoding gene, the expression vector of the present invention can have control antibody chain in host cell and compile The regulation sequence of code gene expression.

Term " regulation sequence " is intended to include that promoter, enhancer and control antibody chain encoding gene are transcribed or translated more Many expression control element (such as polyadenylation signal).The regulation sequence description of the type is in such as Goeddel；Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, CA (1990) in.Skilled artisan would appreciate that including selecting the expression vector design of regulation sequence can be depending on such as waits to turn The factors such as the selection of host cell changed, desired protein expression intensity.Excellent for express in mammalian host cell The regulation sequence of choosing is included in mammalian cell and causes the strong and viral components of composing type protein expression, such as from big and small Cellular virus (CMV) (such as CMV promoter/enhancer), simian virus 40 (SV40) (such as SV40 promoter/enhancer), adenovirus (such as adenovirus major late promoter (AdMLP)) and the promoter of polyoma virus and/or enhancer.For viral regulatory elements And the further describing of sequence, see for example the US 4 of US 5,168,062, the Bell etc. of Stinski, 510,245 Hes The US 4,968,615 of Schaffner etc..

In addition to the gene of encoding antibody chain and regulation sequence, the recombinant expression carrier of the present invention can have extra sequence The sequence (such as origin of replication) replicated in host cell such as those regulation carriers and selectable marker gene.Selectable marker gene Be beneficial to select carrier imported host cell (see for example the US patent No. 4,399,216,4,634,665 and 5 of Axel etc., 179,017).Such as, selectable marker gene general character be the host cell that inserted so that carrier to cytotoxic drug such as G418, Hygromycin or methotrexate have resistance.Preferred selectable marker gene includes dihydrofolate reductase (DHFR) encoding gene (for having the dhfr that methotrexate selects/expands^-Host cell uses) and neo gene (selecting for G418).

In order to express light chain and heavy chain, by standard technique, the expression vector encoding described heavy chain and light chain is transfected into place Chief cell.The various forms that term " transfects " is intended to include multiple being usually used in Exogenous DNA transfered protokaryon or eukaryotic host cell Technology, such as electroporation, calcium phosphate precipitation, DEAE-glucosan transfection etc..Although the most likely at protokaryon or eucaryon Host cell is expressed the antibody of the present invention, but in eukaryotic cell, preferably expresses antibody, particularly thin in mammalian hosts In born of the same parents, this is because compared with prokaryotic cell, correct folding and immunity in above-mentioned eukaryotic cell particularly mammalian cell Active antibodies is assembled and the probability secreted is higher.The prokaryotic expression that it has been reported antibody gene can not produce high yield effectively Active antibodies (Boss, M.A. and Wood, C.R. (1985) Immunology Today 6:12-13).

The mammalian host cell being preferred for expressing the recombinant antibodies of the present invention includes that Chinese hamster ovary celI (includes being described in Urlaub and Chasin, the dhfr in (1980) Proc.Natl.Acad.Sci.USA 77:4216-4220^-Chinese hamster ovary celI, its with Together with DHFR-selected marker as described in R.J.Kaufman with P.A.Sharp (1982) Mol.Biol.159:601-621 Use), NS0 myeloma cell, COS cell and SP2 cell.Move when the recombinant expression carrier of encoding antibody genes is imported suckling Time in thing host cell, by cultivation host cell until antibody is expressed in described host cell, thus produce antibody, or excellent Selection of land, antibody-secreting enters in the culture medium of host cell growth.Then can be by using the method for purifying proteins of standard from cultivation Separation antibody in base.

In order to produce a part such as Fab fragment or the scFv molecule of complete antibody, it is also possible to use host cell.On The deformation stating method is certainly included in the present invention.Such as, with light chain or the heavy chain (but not both) of the antibody of code book invention DNA transfection host cell be probably desirable.If it is not must that the existence of light chain or heavy chain combines for purpose antigen Need, then by utilizing recombinant DNA technology can partially or completely remove the above-mentioned light chain of coding or above-mentioned heavy chain or both DNA. Molecule expressed by the DNA molecular of above-mentioned truncate is similarly included in the antibody of the present invention.Additionally, by the change utilizing standard Method is antibody linked with another kind by the antibody of the present invention, it is possible to produce wherein a heavy chain and a light chain are the present invention Antibody and another heavy chain and another light chain there is the specific pair of merit for the antigen being different from purpose antigen Can antibody.

In the antibody of the recombinant expressed present invention or the preferred system of its antigen-binding portion thereof, it is situated between by calcium phosphate The recombinant expression carrier of encoding antibody heavy and light chain of antibody is imported dhfr by the transfection led^-In Chinese hamster ovary celI.Recombinant expressed at this In carrier, be the most all operably connected cmv enhancer/AdMLP-of the gene of encoding antibody heavy and light chain starts Son controls element so that the transcribing of gene described in potent effect.Recombinant expression carrier also carries DHFR gene, and it is by utilizing Methotrexate secretion/amplification can be used for selecting the dhfr with the transfection of this carrier^-Chinese hamster ovary celI.It is thin that cultivation is chosen for converting host Born of the same parents so that heavy chain of antibody and light chain expression, and separate complete antibody from cultivating basic weight.The molecular biology skill of use standard Art, in order to prepare recombinant expression carrier, transfection host cell, selection transformant, cultivate described host cell and from culture medium Middle acquisition antibody.Therefore, the present invention relates to a kind of host cell by cultivating the present invention in suitable culture medium until this The recombinant antibodies of invention is synthesized, thus the method synthesizing the recombinant antibodies of the present invention.Additionally, the method can include from described Culture medium separates described recombinant antibodies.

As the alternative by phage display screening recombinant antibodies library, can be by other those skilled in the art Known method is for screening big combinatorial library to identify the antibody of the present invention.Substantially, it is possible to use wherein at nucleic acid and Set up close physical linkage between the antibody encoded by it and its antibody characteristic encoded can be relied on for selecting suitable core Any expression system of acid sequence.

In a type of optional expression system, as Szostak and Roberts WO 98/31700 and Roberts, R.W. and Szostak, described in J.W. (1997) Proc.Natl.Acad.Sci.USA 94:12297-12302 , recombinant antibodies library is expressed with the form of RNA-protein fusions.Within the system, its 3 ' end carries puromycin The In Vitro Translation of the synthesis mRNAs of (a kind of peptidyl receptor antibiotic) creates mRNA and its coded peptide or the covalency of albumen Fusions.Therefore, can characteristic based on encoded peptide or albumen (such as antibody or its part) such as described in antibody or as described in its part With the combination of A β (20-42) ball aggressiveness or derivatives thereof, the one concentrating mRNAs complex mixture (such as combinatorial library) is specific mRNA.Encoding antibody or its part the nucleotide sequence that can obtain by screening above-mentioned library can be in the above described manner (as in sucklings In animal host cell) expressed by recombination method, in addition and can by the screening mRNA-peptide fusions of more rounds, Sudden change is imported in the sequence initially selected, or the additive method warp of the external affinity maturation of recombinant antibodies in the above-described manner By further affinity maturation.

The internal combination with in vitro method

The antibody of the present invention can be produced, such as a side likewise by using internal and in vitro method combination In method, A β (20-42) ball aggressiveness or derivatives thereof is first allowed to act on antibody library in host animal body to stimulate A β (20-42) Ball aggressiveness or the generation of derivant binding antibody, realize further antibody followed by one or more ex vivo technique and select And/or Antibody maturation (i.e. optimization).According to an embodiment, such a kind of combined method can include first with described A β (20-42) ball aggressiveness or derivatives thereof immunity inoculation non-human animal (as mice, rat, rabbit, chicken, camel, sheep or goat or it Transgenic variant or gomphosis mouse) to stimulate for the antibody response of antigen, then by utilizing in vivo at described A β (20-42) under ball aggressiveness or derivant effect, the immunoglobulin sequences of the lymphocyte of irriate is prepared and screens phage Display libraries.The first step of this combined method can by above-mentioned relevant with vivo approaches in the way of carry out, and the second step of the method Then can by above-mentioned relevant with in vitro method in the way of carry out.Preferably prepare the lymph from described irriate with in-vitro screening subsequently The method of the phage display library hyperimmune non-human animal of cell includes that those are described by BioSite Inc., sees example Such as WO 98/47343, WO 91/17271, US 5,427,908 and US 5,580,717.

According to another embodiment, a kind of method of combination include first with A β (20-42) the ball aggressiveness of the present invention or Its derivant immunity inoculation non-human animal (as mice, rat, rabbit, chicken, camel, sheep, goat or their gene knockout and/ Or transgenic variant, or gomphosis mouse), and lead in the antibody of described A β (20-42) ball aggressiveness or derivatives thereof with stimuli responsive Crossing screening hybridoma (such as preparing the animal of immunity of hanging oneself) selects generation to have the lymphocyte expecting specific antibody.From Selected clone separates the gene of encoding antibody or single domain antibodies (by using the cloning process such as reverse transcriptase of standard to gather Synthase chain reaction) and stand external affinity maturation, in order to thus improve the binding characteristic of selected antibody.The first of the method Step can by above-mentioned relevant with vivo approaches in the way of carry out, the second step of the method then can be above-mentioned relevant with in vitro method Mode is carried out, and especially by the method utilizing external affinity maturation, such as those are described in WO 97/29131 and WO 00/ Method in 56772.

In further combined method, by using method known to those skilled in the art to be such as chosen for lymph Cell antibody method (SLAM) and be described in (1996) such as US 5,627,052, WO 92/02551 and Babcock, J.S. Method in Proc.Natl.Acad.Sci.USA 93:7843-7848 produces recombinant antibodies from the lymphocyte of single separation. In the method, first with A β (20-42) ball aggressiveness or derivatives thereof vivo immunization inoculation non-human animal (as mice, rat, Rabbit, chicken, camel, sheep, goat or their transgenic variant, or gomphosis mouse) to stimulate for described oligomer or derivative The immunne response of thing, then passes through the individual cells utilizing antigenic specificity haemolytic plaque assay to select secretion purpose antibody.For This, can use the joint of such as biotin that molecules of interest relevant to ball aggressiveness or derivatives thereof or structure is coupled to sheep red carefully Born of the same parents, identify, from there through utilizing haemolytic plaque assay to make it possible to, the individual cells that secretion has suitable specific antibody. After the cell identifying secretion purpose antibody, obtained coding light chain and variable region of heavy chain by reverse transcriptase PCR by cell CDNA, and then in mammalian host cell such as COS or Chinese hamster ovary celI by as described in variable region and suitable immunoglobulin permanent Determine district (such as human constant region) to express together.Then, the cell transfected by expansion, allow with the lymph selected ex vivo expanded The host cell of the immunoglobulin sequences transfection of cell stands further analyzed in vitro and internal selection, such as in order to separate Express the cell of the antibody with binding affinity.Additionally, expanded immunoglobulin sequences can be operated in vitro.

Can select that there is the anti-of required affinity defined herein by performing Dot blot substantially as described above Body.In short, the antigen of serial dilution is invested on solid matrix, preferably it is imprinted on nitrocellulose membrane.Then allow immobilized Antigen contacts with purpose antibody, then passes through and utilizes the second antibody of conjugated enzyme and chrominance response to detect the latter；In regulation Under antibody and antigen concentration, the amount of antibodies provides affinity and measures.Therefore, two kinds of different antibody are for a kind of target Or a kind of antibody is defined herein as the most identical Dot blot bar for the relative affinity of two kinds of different targets Under part, two kinds of antibody-target is used to combine the ratio of the amount of antibody that viewed respective target combines.

Can this just known method obtain the epi-position combined and monoclonal antibody 5F7,10F11,7C6,4B7,6A2, The identical antibody of epi-position that 2F2,4D10,7E5,10C1 or 3B10 combine.

As above-mentioned antibody can compete different target structures, if these structure energy specificitys of at least one reduce Measurable combination of another kind of structure, preferably by the epi-position that offer is overlapping or identical, the most identical epi-position, then at this It is considered as " competition " to specific antibodies.

Rely on they relative affinities to above-mentioned target structure, competition target entity can be used for directly selecting antibody.Therefore, may be used By utilizing competition assay directly to measure relative affinity, the competition entity of form will can be distinguished as different in this algoscopy The competitive structure of labelling contacts with purpose antibody, and releases antibody for these from the relative quantity with these entities of antibodies The relative affinity of each in entity.

By expectation antagonist had the entity of bigger affinity investing on solid matrix carrier and to add appropriate amount (excellent Select molar excess) expectation antagonist there is the competition entity of less affinity to this medium, above-mentioned competition can be used for directly Enrichment has the antibody of the desired relative affinity for target entity.Therefore, expectation parent relatively is shown compared with other antibody Can trend towards being combined with substrate tighter with the antibody of power and can be obtained after the antibody washing less desirably form off, as passed through Wash under low salt concn and then pass through and utilize high salt concentration by the antibody antibody that reversible results of dissociating combine from its target.As Fruit the most if necessary, can carry out several taking turns enrichment.In a particular of the present invention, wherein as in hybridoma or antigen exhibition Show in phage or yeast cells storehouse, by antibody latent gene type and this antibody physical linkage, thus corresponding phenotype can be saved.

In another embodiment of the present invention, employing the Dot blot of improvement, the most immobilized antigen is with molten The entity competition solved and the combination of antibody so that the relative affinity of antibody can be pushed away by the combination percent with immobilized antigen Go out.

Antibody moiety such as Fab and F (ab ')₂Fragment can be by using routine techniques such as with papain or pepsin Digest and be produced from whole antibody.Stick additionally, antibody, antibody moiety and immunity can be obtained by the recombinant DNA technology using standard Attached element molecule.

The invention still further relates to comprise antibody of the present invention and the pharmaceutical preparation (compositions) of the optional carrier being pharmaceutically suitable for. The pharmaceutical composition of the present invention can additionally comprise at least one additional treatment agent, and such as one or more are for discharging the present invention's Antibody is the additional treatment agent of the treatment of useful disease.If, the ball aggressiveness knot of the antibody of the such as present invention and the present invention Close, then pharmaceutical composition can additionally comprise the treatment that one or more are important disease for wherein said ball dimeric active Additional treatment agent.

Pharmaceutically be suitable for carrier include any solvent, disperse medium, coating, antibacterium and antifungal, etc. blend suction Receive delayed-action activator etc., as long as they are physiological compatible.Pharmaceutically acceptable carrier includes such as water, saline, phosphate Buffer saline, glucose, glycerol, ethanol etc., and combinations thereof.In many instances it is preferred that use isotonic agent such as Saccharide, such as mannitol or the polyalcohols of Sorbitol or sodium chloride.The carrier being pharmaceutically suitable for can additionally comprise relatively A small amount of increases antibody half life or auxiliary substance such as wetting agent or emulsifying agent, preservative or the buffer agent of effectiveness.

Such as, pharmaceutical composition can be suitable for parenteral.Here, antibody is preferably prepared as having 0.1- The injectable solution of the antibody content of 250mg/ml.Can prepare injectable solution with liquid or lyophilized form, dosage form is oxygen Change lead glass bottle or phial, ampoule or prefilled syringe.Buffer agent can contain L-Histidine (1-50mM, preferably 5-10mM) and There is 5.0-7.0, the pH of preferably 6.0.More applicable buffer agents include but not limited to sodium succinate, sodium citrate, sodium phosphate Or potassium phosphate buffer agent.Sodium chloride can be used so that the concentration that solution Zhang Du is adjusted to 0-300mM is (for liquid dosage form, excellent Select 150mM).For freeze-dried formulation, may also include cryoprotective agent such as sucrose (such as 0-10%, preferably 0.5-1.0%).Other close Suitable cryoprotective agent is trehalose and lactose.For freeze-dried formulation, may also include filler such as mannitol (such as 1-10%, Preferably 2-4%).Stabilizer such as METHIONINE (such as 51-50mM, preferably 5-10mM) can be used in liquid and freeze-dried formulation.Individual More applicable filleies are glycine and arginine.It is also possible to use surfactant such as polysorbate80 (such as 0-0.05%, Preferably 0.005-0.01%).More surfactant is polysorbate20 and BRIJ surfactant.

The compositions of the present invention can have multiple dosage form.These dosage forms include liquid, semisolid and solid dosage forms, such as liquid Solution (as injectable and can the solution of infusion), dispersion liquid or suspensoid, tablet, pill, powder, liposome and suppository.Preferably Dosage form depend on desired administration fashion and treatment use.Typically, compositions is preferably with injectable or energy infusion Solution form gives, such as, be similar to the compositions of other antibody for people's passive immunization.Preferably route of administration is Parenteral (such as intravenous, subcutaneous, intraperitoneal or intramuscular) is administered.According to a preferred embodiment, by venoclysis or Injection gives antibody.According to another preferred embodiment, give antibody by intramuscular or subcutaneous injection.

Under preparation and holding conditions, therapeutic combination is generally intended to aseptic and stable.Compositions can be formulated as molten Liquid, microemulsion, dispersant, liposome or other be suitable for the ordered structure of high concentration active substance.Can be by requirement be lived Property material (i.e. antibody) introduce in suitable solvent (using a kind of mentioned component or a combination thereof time as required suitably), and then Solution described in aseptic filtration, from preparation sterile injectable solution.Generally by active substance introducing is comprised basic disperse medium Sterile carrier and suitably time other must prepare dispersant in composition.Just it is used for preparing the aseptic of sterile injectable solution to freeze For dry powder, being vacuum dried and spray drying is preferred preparation method, it is created activity by the solution of aseptic filtration before Composition and suitably time more required compositions powder.Can be such as by using the coating of such as lecithin, by dispersant In the case of maintain required particle size or by utilizing surfactant, such that it is able to keep the suitable mobility of solution.Logical Cross and the preparation such as Monostearate and gelatin that postpone absorption are additionally introduced in compositions, it is possible to achieve prolonging of Injectable composition Long absorption.

Although administration fashion preferred for many treatment use is subcutaneous injection, intravenous injection or infusion, the present invention's Antibody can be administered by multiple method known to those skilled in the art.Skilled artisan would appreciate that route of administration and/ Or type depends on desired result.According to specific embodiment, available protection active substance avoids the load quickly discharged Body prepares this active substance, such as, has the formula continuing or controlling release, and it includes implant, transdermal unguentum and microcapsule Embedding release system.The polymer such as ethylene acetate, polyanhydride that biologically degradable biological is compatible, poly-second can be used Alkyd, collagen, polyorthoesters and polylactic acid.The method preparing above-mentioned dosage form is well known to the skilled person；See Such as Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson, compile, Marcel Dekker, Inc., New York, 1978.

According to specific embodiment, the antibody of the present invention can give by per os, for instance in inert diluent or can metabolism In edible carrier.(and more composition also can be encapsulated in hard or soft capsule, tabletted antibody if necessary) Or be directly appended in food.For oral medication, antibody can be mixed with excipient and with oral tablet, buccal tablet, glue The forms such as wafer, elixir, suspensoid, syrup use.If being intended to give the present invention's by being different from parenteral approach Antibody, it may be necessary to select coating from the material stoping this antibody inactivation.

A kind of method that the invention still further relates to ball dimeric active in the individual internal suppression present invention, wherein said individuality suffers from It is directed to the disease of the active particular importance of the ball aggressiveness of amyloid beta protein and the wherein said present invention.In order to suppress anti- The activity of the ball aggressiveness that body combines, described method includes giving individuality by the antibody of at least one present invention.Described individuality is preferred For the mankind.The antibody of the present invention can give human individual for therapeutic purpose.Additionally, the antibody of the present invention can be for veterinary's mesh Or in the animal model framework of disease specific, give non-human mammal.Above-mentioned animal model can be used for evaluating the present invention and resists The therapeutic effect (being such as used for detecting dosage and time-histories) of body.

The disease that wherein the ball aggressiveness of the present invention works includes that wherein its development and/or progress relate to the present invention The disease of ball aggressiveness.The ball aggressiveness of these diseases particularly those wherein present invention be significantly or presumably cause described The reason of the pathophysiology of disease, or it contributes to those diseases of factor of described ongoing disease and/or progress.Cause This, the activity including the ball aggressiveness wherein suppressing the present invention at this can alleviate the symptom of disease and/or those diseases of progress. The concentration that above-mentioned disease can such as be increased by the ball aggressiveness of the present invention in the individual biological fluid suffer from particular condition (concentration as increased in serum, blood plasma, CSF, urine etc.) and is confirmed.This can the antibody of such as the application of the invention obtain Detection.Relevant to the various disease conditions being directed to neural degeneration factor, cognitive defect, neurotoxicity factor and inflammatory factors Pathology in the ball aggressiveness of the present invention played important function.

In another aspect of the present invention, the disease that can be treated or prevent includes that those are relevant to amyloidosis Disease.Term " amyloidosis " herein means many with specific protein (amyloid, threadiness in health different tissues Albumen and their precursor) exception folding, grumeleuse, assemble and/or gather the disease being characterized.In Alzheimer and Tang In Cotard, have impact on nervous tissue, in cerebral amyloid angiopathy (CAA), have impact on blood vessel.

The pharmaceutical composition of the present invention can include the antibody of the present invention of " therapeutically effective amount " or " prevention effective dose " or anti- Body portion." therapeutically effective amount " refers in dosage and the one that can effectively achieve desired therapeutic effect on required a period of time Amount.The therapeutically effective amount of antibody or antibody moiety can be determined by those skilled in the art, and can with such as individual morbid state, Age, sex and body weight and antibody or antibody moiety cause the factor of the ability of expectation response to change in individuality.Treatment The treatment beneficial effect of effective dose or a kind of wherein antibody or antibody moiety exceedes the amount of any toxicity or illeffects." pre- Anti-effective dose " refer in dosage and a kind of amount that desired preventive effect can be effectively achieved on required a period of time.Typically, Owing to before seizure of disease or preventive dose is in early days used for individuality by outbreak, therefore prevention effective dose should less than treatment effectively Amount.

Additionally, the present invention includes a kind of prevention or Alzheimer in the patient of the treatment above-mentioned prevention of needs or treatment Further method.The method includes being enough to the amount of prevention or therapeutical effect and above-mentioned vaccine gives the step of patient.

Additionally, the present invention includes that a kind of qualification is suitable for the trouble that projected extension is amyloidosis such as Alzheimer The method of the compound of person's active immunization.The method includes: 1) one or more purpose compounds are exposed to one or Under conditions of multiple above-mentioned antibody a period of time being in be enough to make these one or more compounds and antibodies；2) identify Those compounds with antibodies, the compound projected extension to be used for identified is amyloidosis such as alzheimer ' Silent sick patient's active immunization.

In the framework that antibody diagnosis uses, qualitatively or quantitatively determining of specificity ball aggressiveness is used in particular for diagnosing the illness Relevant amyloid beta form.Within a context, specificity refer to can with enough sensitivity technique specific ball aggressiveness or its spread out Biology or the probability of its mixture.The antibody of the present invention advantageously has less than 10ng/ml sample, is preferably lower than 1ng/ml Sample and particularly preferably less than the detection threshold concentration of 100pg/ml sample, it is meant that can be examined by the antibody of the present invention Measure the ball aggressiveness concentration in the every milliliter of sample indicated the most in each case, and favourable lower concentration.

Detect according to immunologic method.In principle, it can be by using any analysis which using antibody Or diagnostic measurement method performs, including coagulation and sedimentation, immunoassay, immunohistochemical method and immunoblotting skill Art, such as Western blotting, or preferably dot blotting.Vivo approaches such as imaging method is also included within this.

Application immunoassay are favourable.Competitive immunometric assay (wherein antigen and labelled antigen (tracer) competition Mensuration with antibodies) and sandwich immunoassay (be wherein usually the antibody test of labelling by the second antibody special Property antibody and the mensuration of the combination of antigen) it is all suitable.These mensuration can be that homophase (is not the most separated into solid phase and liquid Phase) or heterogeneous (the most combining labelling is separated with unconjugated labelling, such as, separated by the antibody of solid phase binding). Depending on labelling and assay method, different heterogeneous and homophase immunoassay are divided into specific classification, such as RIAs, and (radiation is exempted from Epidemic disease measure), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescence immunoassay), LIA (luminescent immunoassay), TRFIA (time Between differentiate FIA), IMAC (immune activation mensuration), EMIT (EMIT), TIA (TIA), I- PCR (immuno PCR).

Ball aggressiveness for the present invention is quantitative, and preferably competitive immunometric assay, wherein the ball aggressiveness of the labelling of ormal weight spreads out Biological as tracer with to be competed by the ball aggressiveness of quantitative sample (containing the unlabelled ball aggressiveness of unknown quantity) and to make The combination of antibody.By means of standard curve, can gather from the measure amount of antigen, i.e. ball random sample product of the tracer being replaced The amount of body.

In the labelling that can be used for these purposes, it turned out that enzyme is favourable.Such as can use based on peroxidase special It it not the system of horseradish peroxidase, alkali phosphatase and beta-D-galactosidase.Its transformation can be monitored by photometering Specific substrate be that such as these enzymes are utilizable.For alkali phosphatase, applicable substrate system is based on phosphoric acid pair Nitro phenyl ester (p-NPP), the bromo-4-of 5-chloro-3-indolyl phosphate/nitroblue tetrazolium(BCIP/NPT), fast red/naphthols-AS- TS phosphate ester；For peroxidase, applicable substrate system is based on 2,2-azine group pair (3-ethyl benzo thiazole phenanthroline- 6-sulfonic acid) (ABTS), o-phenylenediamine (OPT), 3,3 ', 5,5 '-tetramethyl benzidine (TMB), 3,3'-dimethoxy-4,4'-diaminobiphenyl., 5-amino water Poplar acid, 3-dimethylaminobenzoic acid (DMAB) and 3-methyl-2-[4-morpholinodithio quinoline hydrazone (MBTH)；For beta-D-galactosidase, Be suitable for substrate system be based on O-Nitrophenylfluorone-β-D-galactoside (o-NPG), p-nitrophenyl-β-D-galactoside and 4-methylumbelliferyl phenyl-β-D-galactoside (MUG).As a rule, these substrate system are with ready-to-use form The most obtainable, such as, with the form of tablet, it also can close the most suitable buffer agent of more reagent etc..

The tracer used can be the ball aggressiveness of labelling.On that point, the ball aggressiveness that labelling is to be determined can be passed through And use it as tracer to measure specific ball aggressiveness.

Can be used for preparing tracer by labelling and ball aggressiveness coupling by per se known manner.By analogize above for The annotation of the ball aggressiveness derivatization of the present invention is also suitable.Additionally, can obtain in a large number for Protein Conjugation and the labelling suitably modified, Such as biotin-conjugated-, avidin-, extravidin-or the enzyme of Succ-PEG-DSPE, maleimide is lived Enzyme changed etc..These labellings can with oligomer or if necessary with the ball aggressiveness direct reaction of suitable derivatization to generate Tracer.Such as, if using Succ-PEG-DSPE-peroxidase conjugated thing, then gather firstly the need of biotinylation ball Body.This is correspondingly applied to contrary order.The method being suitable for this purpose is also road known to those skilled in the art.

If selecting heterogeneous immunoassay format, can be by antigen-antibody complex being combined with carrier and by its point From, such as by separating with the anti-idiotype antibody of described carrier conjugation such as anti-rabbit IgG antibody.The carrier being suitable for, particularly With the coated microtitration plate of suitable antibody be known and part is available commercial.

The invention further relates to that there is at least one antibody described above and more multi-component immunoassay suit.Institute State suit generally to exist with packing unit (combinations of the means that a kind of ball aggressiveness for performing the present invention measures) form.In order to The most easily operating, described means preferably provide with the most ready-to-use form.A kind of favourable configuration is with examination The form of agent box provides immunoassay.Test kit usually contains multiple container for separate configurations component.All of component is all Can be used for the form offer of dilution at any time, it is used for dissolving or weight such as the concentrate for diluting or as dry or lyophilized products Outstanding；Single or all of component can freezing or at room temperature be preserved until using.Serum is preferably such as the coldest at-20 DEG C Freeze (shock-frozen) so that in these cases immunoassay the most sure be preferably maintained at below freezing Temperature.

The more multicomponent being supplied to immunoassay depends on the type of described immunoassay.Generally, by standard protein, can Can need or perhaps without tracer and control serum provide together with antiserum.Additionally, also include microtitration plate (preferred antibody is coated), such as testing, wash or buffer that substrate converts and zymolyte itself.

Immunoassay and be found in example as the antibody producing of auxiliary in laboratory and hospital and the General Principle of use Such as Antibodies, A Laboratory Manual, (Harlow, E., and Lane, D. compile, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Therefore, present invention additionally comprises amyloidosis such as A Erci in a kind of patient diagnosing and suspecting or suffer from this disease The silent sick method in sea.The method comprising the steps of: 1) separates biological sample from patient；2) biological sample is above-mentioned anti-with at least one Under conditions of body contacts a period of time and is in and be enough to form antigen/antibody complex；With 3) detect antigen in described sample/anti- The existence of nanocrystal composition, complex existence indicates the diagnosis of amyloidosis such as Alzheimer in patient.Antigen can To be such as ball aggressiveness or there is its part or the fragment of the functional characteristic identical with complete ball aggressiveness (as combined activity).

Additionally, the present invention includes that another kind of diagnosis is suspected or suffers from amyloidosis such as A Erci in the patient of this disease The silent sick method in sea.The method comprising the steps of: 1) separates biological sample from patient；2) time by biological sample and antigen contact one section Between and under conditions of being in and being enough to form antibody/antigen complex；3) conjugate is added to obtained antibody/antigen complex Under conditions of middle a period of time being in be enough to make conjugate and the antibodies of combination, wherein conjugate comprises has a kind of connection The signal that can produce detectable signal produces the above-mentioned antibody of compound；With 4) produced produced by compound by detection signal The existence of the antibody that signal detection is likely to be present in biological sample, amyloidosis such as A Erci in signal designation patient The silent sick diagnosis in sea.Antigen can be ball aggressiveness or have its of the functional characteristic identical with complete ball aggressiveness (as combined activity) Part or fragment.

The present invention includes that a kind of suspection that diagnoses suffers from amyloidosis in the amyloidosis such as patient of Alzheimer The additional method of sexually transmitted disease (STD) such as Alzheimer.The method comprising the steps of: 1) separates biological sample from patient；2) by biological sample Contact a period of time being in be enough to allow that formation is anti-with anti-antibody (wherein anti-antibody specificity is for a kind of above-mentioned antibody) Under conditions of antibody/antibody complex, this complex contains the antibody being present in biological sample；2) conjugate is added to gained To anti-antibody/antibody complex under conditions of a period of time being in be enough to make the conjugate antibodies with combination, its Middle conjugate comprises the antigen being combined with the signal generation compound that can produce detectable signal；With 3) detection signal generation chemical combination Signal produced by thing, the diagnosis of amyloidosis such as Alzheimer in signal designation patient.

Equally, the present invention includes a kind of test kit, and it comprises: a) at least one above-mentioned antibody, and b) comprises connection and have letter Number producing the conjugate of antibody of compound, wherein the antibody of conjugate is different from the antibody of separation.

Present invention additionally comprises a kind of test kit, it comprises: a) for the anti-antibody of one of above-mentioned antibody, and b) comprises even It is connected to the conjugate that signal produces the antigen of compound.Antigen can be ball aggressiveness or have and this ball aggressiveness identical function characteristic Its fragment of (as combined activity) or part.

In a diagnosis embodiment of the present invention, antibody or its part of the present invention are coated in solid phase (or deposit It is in liquid phase).Then sample or biological sample (such as whole blood, cerebrospinal fluid, serum etc.) are contacted with this solid phase.If antigen (such as ball aggressiveness) is present in sample, and antibody on the most above-mentioned antigen-binding solid phases also then passes through direct or indirect method and carries out Detection.The existence of existence that direct method includes detecting complex self simply and therefore antigen.In indirect method, will Conjugate adds in the antigen of combination.This conjugate comprises and connects the antigen with combination having signal to produce compound or labelling In conjunction with second antibody.Second antibody once combines with the antigen combined, and signal produces compound and just produces measurable signal. The existence of antigen in the most above-mentioned signal designation sample.

Example for the solid phase of diagnostic immunoassay is porous and pore-free material, latex particle, magnetic-particle, microgranule (seeing the U.S. patent No. 5,705,330), pearl, thin film, microtiter well and plastic test tube.If necessary, solid phase material The Performance Characteristics of desired mensuration form is depended in the selection of the method for material and the antigen that is present in conjugate of labelling or antibody.

Signal is had to produce the antibody of compound or labelling (perhaps as it has been described above, conjugate (or indicator) should comprise to connect It is anti-antibody, depends on algoscopy).This signal generation compound or " labelling " are that self is detectable or can be with a kind of or many Plant other compounds to react to produce detectable product.Signal produces the example of compound and includes chromophore, and radioactivity is same Position element (such as 125I, 131I, 32P, 3H, 35S and 14C), chemiluminescence compound are (such as acridine), granule (visible or fluorescence ), nucleic acid, chelating agent or catalyst such as enzyme be (such as alkali phosphatase, acid phosphatase, horseradish peroxidase, beta galactose glycosides Enzyme and ribonuclease).In the case of using enzyme (such as alkali phosphatase or horseradish peroxidase), add colour developing, send The substrate of fluorescence or luminescence can cause the generation of detectable signal.Other detecting systems such as time-resolved fluorometry, internal reflection Fluoremetry, amplification (such as polymerase chain reaction) and Raman spectroscopy are also useful.

Blood plasma, whole blood, dry whole blood, blood can be included by the example of the biofluid that above-mentioned immunoassay are tested Clearly, cerebrospinal fluid or tissue and the water of cell or organic-water extract.

Present invention additionally comprises a kind of for detecting the method that in sample, antibody exists.The method comprising the steps of: (a) be enough to Make time that anti-antibody/antibody complex formed and under the conditions of, by suspect or sample containing antibody and specificity for The anti-antibody contact of antibody in Patient Sample A, during wherein anti-antibody is a kind of combination Patient Sample A, the present invention's of antibody is anti- Body；B () adds conjugate in obtained anti-antibody/antibody complex, this conjugate comprises connection to be had and can detect and can detect The signal of signal produces the antigen (it combines anti-antibody) of compound；(d) produced produced by compound by detection signal The existence of the antibody that signal detection is likely to be present in sample.Comparison or the calibration comprising the antibody for anti-antibody can be used Thing.

Test kit is intended to be included within the scope of the present invention.More specifically, the present invention includes suffering from suspection for mensuration Alzheimer or be characterised by cognitive defect other disease patient in the test kit that exists of antigen (such as ball aggressiveness).Special Not, a kind of test kit existed for measuring antigen in sample comprises a) antibody or its part as herein defined；And b) Comprise and connect the second antibody (there is the specificity for antigen) having the signal that can produce detectable signal to produce compound Conjugate.This test kit also can comprise comparison or the caliberator of the reagent containing being combined with this antigen.

Present invention additionally comprises for detecting the test kit of antibody in sample.This test kit can comprise a) specificity for purpose The anti-antibody (such as one of present invention's) of antibody, and b) antigen as defined above or its part.Comprise and be combined with this antigen The comparison of reagent or caliberator may also comprise wherein.More specifically, this test kit can include that a) specificity is for antibody Anti-antibody (such as one of present invention's), and b) comprise to connect and have the signal that can produce detectable signal to produce compound The conjugate of antigen (such as ball aggressiveness).Additionally, this test kit also can comprise comparison or the school of the reagent containing being combined with this antigen Quasi-thing.

This test kit also can comprise container such as phial, bottle or a silver containing presetting solid phase, and contains Other containers of corresponding conjugate.It is tubular that these test kits also can comprise containing other reagent required for carrying out this mensuration Bottle or container, such as, wash, process and indicator.

Be also noted that the present invention not only includes above-mentioned full length antibody, also include its part or fragment, such as its Fab Part.Additionally, the present invention includes having any anti-of identical characteristics according to such as binding specificity, structure etc. and antibody of the present invention Body.

Advantages of the present invention:

By using A β (20-42) ball aggressiveness immunity inoculation, can obtain by competitive Dot blot as above The toleration that measured or identify different A β (1-42) oligomer and the discrepant different list of A β (X-42) oligomer aspect Clonal antibody.This makes to develop at cognitive potentiation, the desired specificity exceeding other A beta forms and MIN The antibody for the A beta oligomers of N-end truncate between side effect distribution with best relation is able to possibility.Astonishing Ground, A β (1-42) and A β (12-42) is although ball aggressiveness is containing structural detail, but is only by the A β utilizing further truncate (20-42) antibody that ball aggressiveness is obtained as antigen partly identifies.

It is that (the A β ball i.e. using N end truncate gathers basic constituent by specific oligomerization A beta form being limited enzyme action Body), in active immunization, produced antibody repertoire is that high special is in A β oligomerization form.This is also applied for for passively exempting from Epidemic disease inoculates the monoclonal antibody being used.Such a is it for the advantage of the specific policy of immunity inoculation (automatic or passive) The A β peptide or the immunne response of sAPP α for A beta monomers, existed will not be induced with the fibril state assembled.Following side In face, it is favourable:

1) in AD brain with insoluble A β plaque, with assemble fibril state exist A β peptide occupy whole A β peptide storehouse Major part.The excess release being dissolved the A β caused by the reaction induced A β plaque of anti-A β antibody with these speckles is considered as It is harmful to.Then this A β discharged in a large number can pass blood brain barrier, and entering blood flow also increases micro-hemorrhage risk potentially.This Outward, in ELAN tests, due to case outbreak meningoencephalitis, therefore this use fibril shape A β peptide form immunity inoculation of 6% Strategy especially just force this test to be cancelled.

2) immunne response for monomer A β peptide form is undesirable, as it is possible that show that monomer A β peptide form can be sent out Wave cognitive potentiation.

3) immunne response for sAPP α is undesirable equally, it is therefore possible to cause with physiology present on before Body protein APP produces reaction and therefore causes autoimmune response.Additionally, sAPP α also shows the cognitive potentiation of performance.

4) in order to avoid undesirable micro-hemorrhage side effect, it should avoid for the blood vessel A β peptide existed with CAA form Response (when with above-mentioned for time compared with the antibody of N-end, for A β middle body and also not with the A assembled with CAA form Less micro-hemorrhage of antibody induction that beta-peptide combines, sees as above).

5) the A beta substance relevant relative to pathophysiology, can have higher with the antibody of A beta oligomers specific reaction Bioavailability because they will not with as fibril shape A β is combined and therefore cannot obtain therapeutic effect.

Preserved material information: according to budapest treaty clause, produces the hybridoma of monoclonal antibody 5F7 in December, 2005 Within 1st, pay in American type culture collection, 10801 University Boulevard, Manassas, Virginia 20110 preservations, and obtain deposit number PTA-7241.Additionally, according to budapest treaty clause, produce monoclonal antibody The hybridoma of 10F11 is paid in American type culture collection on 1st in December in 2005,10801 University Boulevard, Manassas, Virginia 10801 preservation, and obtain deposit number PTA-7239.It addition, according to Bu Dapei This clause of treaty, the hybridoma producing monoclonal antibody 4B7 is paid in American Type Tissue Culture in December in 2005 on the 1st The heart, 10801 University Boulevard, Manassas, Virginia 10801 preservation, and obtain deposit number PTA- 7242, and according to budapest treaty clause, produce the hybridoma of monoclonal antibody 7C6 in December in 2005 within 1st, pay in American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801 protect Hide, and obtain deposit number PTA-7240.Additionally, according to budapest treaty clause, produce the hybridoma of monoclonal antibody 6A2 Pay in American type culture collection on February 28th, 2006,10801 University Boulevard, Manassas, Virginia 10801 preservation, and obtain deposit number PTA-7409, and according to budapest treaty clause, The hybridoma producing monoclonal antibody 2F2 is paid in American type culture collection on February 28th, 2006, and 10801 University Boulevard, Manassas, Virginia 10801 preservation, and obtain deposit number PTA-7408.According to Budapest treaty clause, the hybridoma producing monoclonal antibody 4D10 was paid in U.S. typical case cultivation on February 28th, 2006 Thing preservation center, 10801 University Boulevard, Manassas, Virginia 10801 preservation, and obtain preservation Numbering PTA-7405.According to budapest treaty clause, the hybridoma producing monoclonal antibody 7E5 was handed on August 16th, 2006 Pay in American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801 Preservation, and obtain deposit number PTA-7809.According to budapest treaty clause, produce the hybridoma of monoclonal antibody 10C1 in On August 16th, 2006 pays in American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801 preservation, and obtain deposit number PTA-7810.According to budapest treaty clause, produce The hybridoma of monoclonal antibody 3B10 is paid in American type culture collection on 1st in JIUYUE in 2006, and 10801 University Boulevard, Manassas, Virginia 10801 preservation, and obtain deposit number PTA-7851.All Preservation be all with Alpert (Abbott) laboratory, 100 Abbott Park Road, Abbott Park, Illinois The name of 60064 (US) is carried out.

Accompanying drawing explanation

In the accompanying drawings:

Fig. 1 shows A β (1-42) and the size exclusion chromatography of A β (1-40).A β (1-42) monomer is dissolved in A) 0.1% NH₄OH, B) 70% formic acid, C) in 0.1%NaOH and D) A β (1-40) is dissolved in 0.1%NaOH.Subsequently, sample is at 20mM NaH₂PO₄, 140mM NaCl, pH 7.4 dilutes further with 1: 10.After at room temperature dissolving, these samples of incubation 5 minutes (left hurdle) or 1 hour (right hurdle), is then applied to size exclusion pillar；

Fig. 2 A) show standard protein (biomarker proteins proteins, swimming lane 1)；A β (1-42) fibril goods, compare (swimming lane 2)；A β (1-42) fibril goods+mAb 5F7,20h, 37 DEG C, supernatant (swimming lane 3)；A β (1-42) fibril goods+mAb 5F7,20h, 37 DEG C, precipitate (swimming lane 4)；A β (1-42) fibril goods+mAb 6E10,20h, 37 DEG C, supernatant (swimming lane 5)；A β (1-42) fibril goods+mAb 6E10,20h, 37 DEG C, the SDS PAGE of precipitation (swimming lane 6)；

B) show that being combined mAbs with A β-fibril accounts for the quantitative analysis results of total antibody percent；

Fig. 3 is a column diagram, which show the APP/L mice processed with wild-type mice (positive control) and PBS- (negative control) is compared, with being dissolved in 0.1%NH₄A β (1-42) monomer, A β (1-42) ball aggressiveness and A β (20-42) ball in OH After aggressiveness active immunization, use the result of the object identification test of APP/L transgenic mice, wherein according to P in ANOVA To the inspection of t-afterwards between different groups after ＜ 0.05, circle represents the APP/L mice significant difference processing PBS-, asterisk table Show Random Level (50%) highly significant difference；

Fig. 4 shows 100pmol/ μ l (A row)；10pmol/ μ l (B row)；1pmol/ μ l (C row)；0.1pmol/ μ l (D row) A β (1-42) ball aggressiveness (the 1st hurdle) with 0.01pmol/ μ l (E row)；The A β of the HFIP pretreatment being dissolved in Pluronic F68 (1-42) monomer (the 2nd hurdle)；A β (20-42) ball aggressiveness (the 3rd hurdle)；A β (12-42) ball aggressiveness (the 4th hurdle)；It is dissolved in DMSO A β (1-40) monomer (the 5th hurdle) of HFIP pretreatment；A β (1-42) monomer, NH₄OH (the 6th hurdle)；A β (1-42) fibril goods (the 7th hurdle) and from Sigma sAPP α (the 8th hurdle) with after A β (20-42) ball aggressiveness active immunization APP/L Tg mice The Dot blot of the sero-fast reactivity of difference obtained；

Fig. 5 is a column diagram, which show with A β (1-42) monomer (0.1%NH₄OH), A β (1-42) ball aggressiveness, A β (20-42) ball aggressiveness or as comparison carrier active immunization APP/PS1 Tg-mice brain extract in solubility With insoluble A β (1-42) and the concentration of A β (1-40) peptide；

Fig. 6 is a column diagram, which show compared with control mice, with anti-A β (20-42) ball dimer antibody 5F7, The result of the object identification test carried out with APP/L transgenic mice after 10F11 and 7C6 passive immunization, A) it is respectively directed to Every kind of antibody and B) all antibody consider in the lump；

Fig. 7 A) show different anti-A β antibody (-6E10 ,-5F7 ,-4B7 ,-10F11 ,-6A2 ,-4D10 ,-3B10 ,- 2F2 ,-7C6 ,-7E5 ,-10C1) specific dot blot assay.Monoclonal antibody at this acceptance test is by with A β (20-42) ball aggressiveness active immunization mice, the hybridoma acquisition merged by selection subsequently (except commercially available 6E10, Outside Signet numbering 9320).By single A beta form serial dilution and be used for immunoreation with corresponding monoclonal antibody incubation:

1.A β (1-42) monomer, 0.1%NH₄OH

2.A β (1-40) monomer, 0.1%NH₄OH

3.A β (1-42) monomer, 0.1%NaOH

4.A β (1-40) monomer, 0.1%NaOH

5.A β (1-42) ball aggressiveness

6.A β (12-42) ball aggressiveness

7.A β (20-42) ball aggressiveness

8.A β (1-42) fibril goods

9.sAPP α (Sigma) (the first speckle: 1pmol)

B) photodensitometry is used to carry out quantitative assessment.For every kind of A beta form, only corresponding to minimum antigen concentration Speckle is evaluated, and precondition is that it has relatively in the phase of final optically clear A β (20-42) the ball aggressiveness speckle identified Relative density to density (threshold value) big 20%.Each Dot blot is independently measured this threshold value.This value indicates for given Antibody is in the relation identified between A β (20-42) ball aggressiveness and corresponding A beta form；

Fig. 8 shows antibody Ahl tribulus sea silent sickness under variable concentrations (, AD) patient or old APP transgenic mice The combination of the slices across of neopallium:

A) Amyloid deposits is verified by congo red staining, APP transgenic mouse lines Tg2576 and AD patient (RZ55) as speckle in cerebral tissue, as cerebral amyloid angiopathy (CAA) in cerebrovascular；

B) in AD patient (RZ16), the strong dyeing of A β (amyloid speckle) substantial precipitation occurs over just use 6G1 and city In the case of selling antibody 6E10 (left hurdle), and antibody 5F7,2F2 and 6A2 (the second hurdle), 4D10,10F11 and 3B10 (third column) And 7C6,7E5 and 10C1 (right hurdle) the most do not show dyeing.All antibody all use under 0.7 μ g/ml concentration；

C) in 19 months big Tg2576, the strong dyeing of A β (amyloid speckle) substantial precipitation occurs over just use In the case of 6G1 and commercial antibody 6E10 (left hurdle), and antibody 5F7,2F2 and 6A2 (the second hurdle), 4D10,10F11 and 3B10 (third column) and 7C6,7E5 and 10C1 (right hurdle) the most do not show dyeing.All antibody all make under 0.7 μ g/ml concentration With；

D)-G) use graphical analysis, A β plaque dyeing in histology picture is carried out quantitative analysis.By by the gray scale of speckle The gray value of value subtracting background tissue calculates optical density value (0%=dye-free): D) at 0.7 μ g/ in old Tg2576 mice Under ml antibody dye, E) in APP/L mice under the antibody of 3 kinds of variable concentrations dye, F) in AD patient (RZ55) 0.7 Dye under μ g/ml antibody, and G) dye under the antibody of 3 kinds of variable concentrations in AD patient (RZ16).To commercial antibody 6E10 (asterisk) and 4G8 (circle) and every other antibody (three asterisks/circle: relative to compareing p ＜ 0.001；Thing after ANOVA Rear Bonferroni ' s t-verifies as p ＜ 0.001) dyeing between difference carry out statistical evaluation (D, F).At E) and G) In, compared with commercial antibody 6E10 and 4G8, all antibody in addition to 6G1 always show that notable less dyeing is (at ANOVA After middle p ＜ 0.001, p ＜ 0.001 in t-afterwards checks).

H) the strong dyeing (arrow) of A β blood vessel deposit occurs only at 6G1 and commercial antibody 6E10 (left hurdle), and antibody 5F7,2F2 and 6A2 (the second hurdle), 4D10,10F11 and 3B10 (third column) and 7C6,7E5 and 10Cl (right hurdle) all show nothing Dyeing.All antibody all use under 0.7 μ g/ml concentration.Analogue sees Tg2576 mice (not showing at this) qualitatively；

After Fig. 9 active immunization about 1 year, the anti-A β-antibody titer in TG2576 mice plasma and Dot blot select Property spectrum.Evaluation A) A β (20-42) ball aggressiveness, B) A β (12-42) ball aggressiveness, C) A β (1-42) monomer and D) carrier is last The anti-A β antibody of immunity inoculation plasma sample of Tg2576-mice after about 1 year produces and is indicated also by Dot blot.

1.A β (1-42) ball aggressiveness

2.A β (1-42) monomer, HFIP pretreatment, it is dissolved in 0.1%Pluronic F68

3.A β (20-42) ball aggressiveness

4.A β (12-42) ball aggressiveness

5.A β (1-40) monomer, HFIP pretreatment, 5mM DMSO solution

6.A β (1-42) monomer, 0.1%NH₄OH

7.A β (1-42) fibril goods

8.sAPPα(Sigma)；(the first speckle: 1pmol)；

Figure 10 shows to sum up suffer from the people of Alzheimer and gather without A β (20-42) ball in the cerebral tissue of dementia comparison The form of body level；

Figure 11 illustrates the heavy chain of following monoclonal antibody (mAbs) and the nucleotide of variable region of light chain and aminoacid Sequence (in every aminoacid sequence, complementary determining region (CDRs) underlines).

Following example are intended to illustrate the present invention, and do not limit its scope.

Detailed description of the invention

Embodiment 1: the preparation of ball aggressiveness

A) A β (1-42) ball aggressiveness

A β (1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) is suspended in 6mg/mL In 100%1,1,1,3,3,3-hexafluoros-2-propanol (HFIP), and at 37 DEG C incubated under agitation 1.5 hours to be completely dissolved.HFIP As hydrogen bond rupture agent and for eliminating the structural heterogeneity being pre-existing in A β peptide.Removed by evaporation in SpeedVac HFIP, and A β (1-42) is resuspended in dimethyl sulfoxide under 5mM concentration also supersound process 20 seconds.At phosphate buffered saline (PBS) (PBS)(20mM NaH₂PO₄, 140mM NaCl, pH 7.4) in the A β (1-42) of HFIP pretreatment is diluted to 400 μMs and adds 2% sodium lauryl sulphate (SDS) (aqueous solution) (final concentration of 0.2%SDS) of 1/10 volume.Incubation 6 hours at 37 DEG C Create 16/20-kDa A β (1-42) ball aggressiveness (short-form of spherical oligomer) intermediate.By the H with 3 volumes₂O enters one Step dilutes and incubation creates 38/48-kDa A β (1-42) ball aggressiveness for 18 hours at 37 DEG C.It is centrifuged 20 minutes under 3000g After, by ultrafiltration (30-kDa molecular cut off) concentrating sample, to 5mM NaH₂PO₄, 35mM NaCl, pH 7.4 dialyses, It is centrifuged 10 minutes under 10000g, and takes out the supernatant comprising 38/48-kDa A β (1-42) ball aggressiveness.Alternative as dialyse Scheme, also by 4 DEG C with 9 times of excess (v/v) ice cold methanol/acetic acid solution (33% methanol, 4% acetic acid) precipitation 38/48-kDa A β (1-42) ball aggressiveness 1 hour.Then precipitation 38/48-kDa A β (1-42) ball aggressiveness (under 16200g 10 points Clock), it is resuspended in 5mM NaH₂PO₄, in 35mM NaCl, pH 7.4, and pH is adjusted to 7.4.

B) A β (1-42) the ball aggressiveness cross-linked

A β (1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) is suspended in 6mg/mL In 100%1,1,1,3,3,3-hexafluoros-2-propanol (HFIP), and at 37 DEG C incubated under agitation 1.5 hours to being completely dissolved.HFIP As hydrogen bond rupture agent and for eliminating the structural heterogeneity being pre-existing in A β peptide.Removed by evaporation in SpeedVac HFIP, and A β (1-42) is resuspended in dimethyl sulfoxide under 5mM concentration also supersound process 20 seconds.At phosphate buffered saline (PBS) (PBS)(20mM NaH₂PO₄, 140mM NaCl, pH 7.4) in the A β (1-42) of HFIP pretreatment is diluted to 400 μMs and adds 2% sodium lauryl sulphate (SDS) (aqueous solution) (final concentration of 0.2%SDS) of 1/10 volume.Incubation 6 hours at 37 DEG C Create 16/20-kDa A β (1-42) ball aggressiveness (short-form of spherical oligomer) intermediate.By the H with 3 volumes₂O enters one Step dilutes and incubation creates 38/48-kDa A β (1-42) ball aggressiveness for 18 hours at 37 DEG C.Now by with 1mM glutaraldehyde Incubation 2 hours under 21 DEG C of room temperatures (RT), then processes 30 minutes with ethanolamine (5mM) under RT and carries out 38/48-kDa A β (1-42) crosslinking of ball aggressiveness.

C) A β (20-42) ball aggressiveness

By 1.59ml A β (1-42) the ball aggressiveness goods prepared according to embodiment 1a and 38ml buffer (50mM MES/ NaOH, pH 7.4) and 200 μ l 1mg/ml thermolysin (Roche) aqueous solution.Reactant mixture is stirred under RT 20 hours.Then add 80 μ l 100mM EDTA aqueous solutions, pH 7.4, and will mix with the SDS solution of 1% concentration of 400 μ l Thing is adjusted to the SDS content of 0.01% further.By 15ml 30kDa Centriprep pipe, reactant mixture is concentrated into about 1ml.Concentrate and 9ml buffer (50mM MES/NaOH, 0.02%SDS, pH 7.4) are mixed and be again concentrated to 1ml.? 11 buffer (5mM sodium phosphate, 35mM NaCl) are dialysed 16 hours at 6 DEG C by Dialysis tubing by concentrate.By 2% concentration SDS aqueous solution is by the SDS content of dialysis solution regulation to 0.1%.Centrifuge A sample 10 minutes take out A β (20-42) under 10000g Ball aggressiveness supernatant.

D) A β (12-42) ball aggressiveness

By 2ml A β (1-42) the ball aggressiveness goods prepared according to embodiment 1a and 38ml buffer (5mM sodium phosphate, 35mM Sodium chloride, pH 7.4) and 150 μ l 1mg/ml GluC endo protease (Roche) aqueous solution.Under RT, stirring reaction is mixed After compound 6 male servant, then add 150 other μ l 1mg/ml GluC endo protease (Roche) aqueous solutions.Stir under RT Reactant mixture 16 hours again, then adds 8 μ l 5M DIFP solution.Will reaction by 15ml 30kDa Centriprep pipe Mixture is concentrated into about 1ml.By the mixing of concentrate and 9ml buffer (5mM sodium phosphate, 35mM sodium chloride, pH 7.4) and again Secondary it is concentrated into 1ml.1l buffer (5mM sodium phosphate, 35mM NaCl) is dialysed at 6 DEG C by Dialysis tubing by concentrate 16 little Time.With the SDS aqueous solution of 1% concentration by the SDS content of dialysis solution regulation to 0.1%.Centrifuge A sample 10 minutes under 10000g And take out A β (12-42) ball aggressiveness supernatant.

Embodiment 2: different A β (1-42) monomers and the size exclusion chromatography of A β (1-40) monomer goods

A β (1-42), 0.1%NH₄OH:

1mg A β (1-42) (Bachem, catalog number H-1368) is dissolved in 500 μ l 0.1%NH₄In OH aqueous solution And it is stirred at room temperature 1 minute.Centrifuge A sample 5 minutes under 10 ' 000g.Collect supernatant.According to Bradford ' s method (BIO- RAD) A β (1-42) concentration in supernatant is measured.

5 minutes samples:

Use 20mM NaH₂PO₄, 20 μ l are dissolved in containing 0.1%NH by 140mM NaCl, pH 7.4₄A β in the supernatant of OH (1-42) A β (1-42) concentration of 0.2mg/ml it is diluted to.At room temperature incubated samples 5 minutes.Then size exclusion chromatography is passed through (SEC) 100 μ l are analyzed.

1 hour sample:

Use 20mM NaH₂PO₄, 20 μ l are dissolved in containing 0.1%NH by 140mM NaCl, pH 7.4₄A β in the supernatant of OH (1-42) A β (1-42) concentration of 0.2mg/ml it is diluted to.At room temperature incubated samples 1 hour.Then size exclusion chromatography is passed through (SEC) 100 μ l are analyzed.

A β (1-42), 70%HCOOH:

1mg A β (1-42) it is dissolved in 50 μ l 70%HCOOH aqueous solutions and is stirred at room temperature 1 minute.10 ' Centrifuge A sample 5 minutes under 000g.Collect supernatant.A β (the 1-in supernatant is measured according to Bradford ' s method (BIO-RAD) 42) concentration.

5 minutes samples:

Use 20mM NaH₂PO₄, the A β (1-42) that 2 μ l are dissolved in 70%HCOOH by 140mM NaCl, pH 7.4 is diluted to A β (1-42) concentration of 0.2mg/ml is also adjusted to pH 7.4 with 1M NaOH.At room temperature incubated samples 5 minutes.Then by big Little exclusion chromatographic analysis 100 μ l.

1 hour sample:

Use 20mM NaH₂PO₄, the A β (1-42) that 2 μ l are dissolved in 70%HCOOH by 140mM NaCl, pH 7.4 is diluted to A β (1-42) concentration of 0.2mg/ml is also adjusted to pH 7.4 with 1M NaOH.At room temperature incubated samples 1 hour.Then by big Little exclusion chromatographic analysis 100 μ l.

A β (1-42), 0.1%NaOH:

1mg A β (1-42) (Bachem, catalog number H-1368) is dissolved in 500 μ l 0.1%NaOH aqueous solutions And it is stirred at room temperature 1 minute.Centrifuge A sample 5 minutes under 10 ' 000g.Collect supernatant.According to Bradford ' s method (BIO- RAD) A β (1-42) concentration in supernatant is measured.

5 minutes samples:

Use 20mM NaH₂PO₄, the A β (1-42) that 20 μ l are dissolved in 0.1%NaOH by 140mM NaCl, pH 7.4 is diluted to The concentration of 0.2mg/ml A β (1-42).At room temperature incubated samples 5 minutes.Then 100 μ l are analyzed by size exclusion chromatography.

1 hour sample:

Use 20mM NaH₂PO₄, the A β (1-42) that 20 μ l are dissolved in 0.1%NaOH by 140mM NaCl, pH 7.4 is diluted to The concentration of 0.2mg/ml A β (1-42).At room temperature incubated samples 1 hour.Then 100 μ l are analyzed by size exclusion chromatography.

A β (1-40), 0.1%NaOH:

1mg A β (1-40) (Bachem, catalog number H-1194) is dissolved in 500 μ l 0.1%NaOH aqueous solutions And it is stirred at room temperature 1 minute.Centrifuge A sample 5 minutes under 10 ' 000g.Collect supernatant.According to Bradford ' s method (BIO- RAD) A β (1-42) concentration in supernatant is measured.

5 minutes samples:

Use 20mM NaH₂PO₄, the A β (1-40) that 20 μ l are dissolved in 0.1%NaOH by 140mM NaCl, pH 7.4 is diluted to The concentration of 0.2mg/ml A β (1-40).At room temperature incubated samples 5 minutes.Then 100 μ l are analyzed by size exclusion chromatography.

1 hour sample:

Use 20mM NaH₂PO₄, the A β (1-40) that 20 μ l are dissolved in 0.1%NaOH by 140mM NaCl, pH 7.4 is diluted to The concentration of 0.2mg/ml A β (1-40).At room temperature incubated samples 1 hour.Then 100 μ l are analyzed by size exclusion chromatography.

Condition for size exclusion chromatography (SEC):

SEC post: Superose 12 HR 10/300 GL (Amersham, catalog number 17-5173-01)

Flow velocity: 0.5ml/min

Paper feeding: 0.2cm/min

Trap at 214nm: 0-0.2 absorbance units

Flowing phase: 20mM NaH₂PO₄, 140mM NaCl, pH 7.4

Result is shown in Figure 1.

Owing to A β peptide especially A β (1-42) monomer strong tendency becomes fibril in gathering, therefore prepare monomer purely A β-solution is a huge challenge job.While it is true, in order to screen and identify differentiation A β (1-42)-monomer and A β (1- 40) anti-A β (20-42) the ball aggressiveness of-monomer, just should use best technology to obtain A β-monomer goods.Here test At 20mM NaH₂PO₄, after 140mM NaCl, pH 7.4 dilutes further, the initial solvent of the A β peptide work to building-up effect With.A β peptide supplier (Bachem) states that in their technical data A β (1-42) should be dissolved in 0.1%NH₄In OH.At A β (1-42) in middle NH₄OH dissolves and immediately at 20mM NaH₂PO₄, in 140mM NaCl, pH 7.4 after further 1: 10 dilution, Lower 5 minutes of room temperature (RT), size exclusion chromatography shows that the A β (1-42) at 74kD with small peak is collected as fibril precursor First sign.Monomer A β (1-42) flows out at 11kD main peak and 6kD shoulder.Under room temperature (RT), incubation is after 1 hour, molten In NH₄A β (1-42) peptide high aggregation in OH is A β (1-42) fibril, causes loss not enter into size exclusion chromatography The detectable material of post.If using 70% formic acid as the initial solvent of A β (1-42) peptide, then sent out after 1 hour under RT Raw high aggregation only makes A β (1-42) monomer of smaller portions stay and (notices that formic acid self causes at Protein Detection wavelength High background absorption).The best initial solvent stoping gathering for A β (1-42) is 0.1%NaOH, and it is even the temperature of 1 hour After educating all or solution, and dilution display only has the A β (1-42) of gathering of smaller portions further, and most A β (1-42) remains monomer.The A β (1-40) being initially dissolved in 0.1%NaOH even under RT incubation after 1 hour the most completely Do not show the sign of gathering.

Embodiment 3: by A β (20-42) ball aggressiveness antibodies selective for the SDS-of the differentiation of A β (1-42) fibril PAGE visualizes semi-quantitative analysis

A β (1-42) fibril goods:

At room temperature, 1mg A β (1-42) (Bachem, catalog number: H-1368) is dissolved in 500 μ l 0.1% NH₄In OH aqueous solution and stir 1 minute.Centrifuge A sample 5 minutes under 10 ' 000g.Collect supernatant.According to Bradford ' s method (BIO-RAD) A β (1-42) concentration in supernatant is measured.

100 μ l are dissolved in 0.1%NH₄A β (1-42) in OH and 300 μ l 20mM NaH₂PO₄, 140mM NaCl, pH 7.4 mixing are also adjusted to pH 7.4 with 2%HCl.Then incubated samples 20 hours at 37 DEG C.It is centrifuged under 10 ' 000g at sample After 10 minutes.Abandoning supernatant, and by residue and 400 μ l 20mM NaH₂PO₄, 140mM NaCl, pH 7.4 mixes, all Cross vigorous agitation (" vortex ") 1 minute resuspended and under 10 ' 000g centrifugal 10 minutes.Abandoning supernatant, and by residue and 400 μl 20mM NaH₂PO₄, 140mM NaCl, pH 7.4 mixes, resuspended again by vigorous agitation (" vortex ") 1 minute and 10 ' It is centrifuged 10 minutes under 000g.Abandoning supernatant.Residue is resuspended in 380 μ l 20mM NaH₂PO₄, 140mM NaCl, pH 7.4 In and promote resuspended by vigorous agitation (" vortex ").

Anti-A β antibody and the combination of A β (1-42) fibril:

With 160 μ l 20mM NaH₂PO₄, 140mM NaCl, 0.05%Tween 20, pH 7.4 dilutes 40 μ l A β (1-42) Fibril goods are also stirred at room temperature 5 minutes, then Centrifuge A sample 10 minutes under 10 ' 000g.Abandoning supernatant, and by residual Thing is stayed to be resuspended in 95 μ l 20mM NaH₂PO₄, in 140mM NaCl, 0.05%Tween 20, pH 7.4.Pass through vigorous agitation (" vortex ") promotes resuspended.

The fibril goods aliquot of 10 μ l is each mixed with following:

a)10μl 20mM NaH₂PO₄, 140mM NaCl, pH 7.4

B) 5F7 of 10 μ l 0.5 μ g/ μ l, is dissolved in 20mM NaH₂PO₄, in 140mM NaCl, pH 7.4

C) 6E10 (Signet Nr.:9320) of 10 μ l 0.5 μ g/ μ l, is dissolved in 20mM NaH₂PO₄, 140mM NaCl, pH In 7.4

Incubated samples 20 hours at 37 DEG C, are then centrifuged 10 minutes under 10 ' 000g.Collect supernatant and with 20 μ l SDS-PAGE sample buffer mixes.By residue and 50 μ l 20mM NaH₂PO₄, 140mM NaCl, 0.025%Tween 20, It is resuspended that pH 7.4 mixes merga pass " vortex ", then Centrifuge A sample 10 minutes under 10 ' 000g.Abandoning supernatant, and will residual Thing and 20 μ l 20mM NaH₂PO₄, 140mM NaCl, 0.025%Tween 20, pH 7.4 mixes, then with 20 μ l SDS- PAGE sample buffer mixes.Apply a sample to carry out in 4-20%Tris/ glycine gels gel electrophoresis.

SDS-PAGE parameter:

SDS sample buffer: 0.3g SDS

4ml 1M Tris/HCl pH 6.8

8ml glycerol

1ml 1% bromophenol blue ethanol solution

Use H₂O is filled to 50ml

4-20%Tris/ glycine gels: (Invitrogen, catalog number: EC6025BOX)

Electrophoretic buffer: 7.5g Tris

36g glycine

2.5g SDS

Use H₂O is filled to 2.51

Gel is run under the constant current of 20mA.

Gel-colored: Coomassie blue R250

Result is shown in Figure 2.

The semi-quantitative analysis of different anti-A β antibody and the differentiation of their A β (1-42) fibril.Antibody location, A β (1-42) fibril and A β (1-42) monomer tag are at gel edges.In view of their size, A β (1-42) fibril cannot enter Enter PAGE gel and can be seen in gel groove.

1. labelling

2.A β (1-42) fibril goods；Comparison

3.A β (1-42) fibril goods；+mAb 5F7；20h 37℃；Supernatant

4.A β (1-42) fibril goods；+mAb 5F7；20h 37℃；Precipitation

5.A β (1-42) fibril goods；+mAb 6E10；20h 37℃；Supernatant

6.A β (1-42) fibril goods；+mAb 6E10；20h 37℃；Precipitation

By measuring the optical density of heavy chain of antibody in (pellet fraction) and supernatant fraction that fibril combines after centrifugal (OD) value, is combined by SDS-PAGE assay is relative with fibril d type A β.The antibody being combined with A β fibril should with A β- Therefore fibril co-precipitation also sees in pellet fraction, and (dissociating) antibody that non-A β-fibril combines then sees supernatant Liquid.Percent according to the antibody that equation below calculating is combined with A β-fibril:

The percent of the antibody being combined with A β-fibril=

OD_{Fibril fraction}X100%/(OD_{Fibril fraction}+OD_{Supernatant fraction}).

To mAbs 6E10 (Signet, catalog number: 9320), 5F7,2F2,6A2,4D10,10F11,3B10,7C6, 7E5 and 10C1 carries out this operation.

In Alzheimer brain, A β fibril is the main component in total A β peptide storehouse.By with anti-A β-antibodies attack These fibrils, owing to discharging a large amount of A β, it can increase micro-hemorrhage danger subsequently, therefore increases the danger of reverse side side effect Danger.With the micro-hemorrhage risk (Bennett that observed increase in the fibril aggregation active immunization method of A β peptide And Holtzman, 2005, Neurology, 64,10-12；Orgogozo J, Neurology, 2003,61,46-54；Schenk Deng, 2004, Curr Opin Immunol, 16,599-606).

And commercial antibody 6E10 (Signet 9320) contrast of the raw A β-epi-position of the line identified between AA1-17, in co-precipitation In experiment, A β (20-42) ball aggressiveness antibodies selective 5F7 is (compared with other A β-forms, for A β (20-42) ball aggressiveness in fact There is on border minimum selectivity) be not combined with A β (1-42) fibril.This demonstrate in supernatant after settling step, with After A β (1-42) fibril incubation, 5F7 antibody still retains and is not co-precipitated (co-precipitation is because being combined with A β (1-42) fibril) The fact.

Embodiment 4: compared with wild type, with A β (1-42) monomer (0.1%NH₄OH), A β (1-42) ball aggressiveness or A β (20-42) by utilizing the cognitive performance of object identification test analysis mice after the active immunization of ball aggressiveness

In these experiments, the process LAN mice with the people APP of point mutation is employed.This point mutation refers to aminoacid 717 (isoleucine for valine) has also seen in London (London) family, and in this family, it caused AD to show effect before 60 years old (Mullan etc., Nature Genetics 2 (1992) 340-342).Leuven creates and describes first referred to herein as The transgenic mice (Moechars etc., J.Biol.Chem.274 (1999) 6483-6492) of APP/L.When 6 week old to female APP/L mice carries out active immunization.

The 100 μ g that in mouse peritoneum, acceptance mixes with equivalent complete Freund's adjuvant are dissolved in phosphate buffered saline (PBS) (PBS) In A β (1-42) monomer (0.1%NH₄OH), A β (1-42) ball aggressiveness or A β (20-42) ball aggressiveness, then every three weeks use Same amount of antigen booster injection in incomplete Freund's adjuvant, continue three months.In whole experimentation, contrary Animal is remained at the condition of standard by day night circulation (14 hours illumination/10 hour starting from 7 in afternoon are dark) Under.Past along with experimental period, it is contemplated that body weight increases and as broad as long with the matched group individually accepting PBS/ adjuvant, hint Antigen process has well tolerable.

By object identification test verification mice the most described in the art at the cognitive competence at 4.5 monthly ages (Journal of Neuroscience 22 (2002) 3445-3453 such as Dewachter).To this end, allow mice get used to activity Place also is then exposed to detect 10 minutes (acquisition) stages, they is individually placed in during this period and now contains two In the playground of similar elements (similarly sized for about 4cm blue cone, green cubic body, yellow cylinder).Record little The persistent period of Mus detecting object and frequency.During memory stage, after 2.5 hours, mice is sent back to except known object it In the outer existing playground containing the random unknown object being selected from other objects.Identification record to new object is relative to time total Between (detecting old and new object) mice detection past heritage body during time." discrimination index (recognition Index) " have expressed this relation (for the time/total time of new object).The mice not remembeing known object is considered as New object is also interested in and the time detection of spending same by it, in other words, has the discrimination index of 50%.Remember known The mice of object is considered as different interested and therefore has considerably higher discrimination index.Known APP/L mice exists 4.5 monthly ages were cognitive defects, and had the discrimination index in Random Level (i.e. 50%) yardstick.

Result is shown in Figure 3.

Object identification test is carried out in mice.This test report is according to the detection row during 10 minutes test phases By the identification with unknown object phase comparison known object measured.Discrimination index is defined relative to spend in two kinds of things of detection Time on body, mice spent percentage of time on detection unknown object.At test phase first 3 hours, visited at 10 minutes Known object is exposed to mice during the stage by survey.Compare five groups of mices and (under bar post, give numbering n).Normally C57B1/6 mice (wild type) display and Random Level (50%, i.e. spend in known equal with detection time on unknown object) High RI significant difference (* * *=p ＜ 0.001；Student t-test).Other four groups of APP transgenic mice warps before three months Go through active immunization.The immunogen used is that A β (1-42) monomer, A β (1-42) ball aggressiveness and A β (20-42) ball are poly- Body.Phosphate buffered saline (PBS) (PBS) is with comparing.Significant difference between PBS and other groups is expressed as circle:^○=p ＜ 0.05；^○○=p ＜ 0.01 (in ANOVA after p ＜ 0.05, t-inspection afterwards).

Compared with nontransgenic mice, it is known that APP/L mice shows cognitive defect when 4.5 monthly age, and appraisal result connects It is bordering on Random Level (i.e. 50% discrimination index).It is true that compared with nontransgenic mice (wild type), the mice that PBS processes Show random behavior.By natural A β (1-42) ball aggressiveness and A β (20-42) ball aggressiveness immunity inoculation in APP/L mice Produce the object identification significantly improved.

Because two kinds of ball aggressiveness goods (natural and truncate) all create memory improvement, very in APP transgenic animal To creating the best identification in the animal processed with A β (20-42) ball aggressiveness, therefore have reason to estimate for cutting The antibody induction of short A β (20-42) ball aggressiveness can produce best result, and use has atopic with this material Antibody carry out passive immunization and represent the optimal strategy for the treatment of.

Embodiment 5: with after A β (20-42) ball aggressiveness active immunization APP/L Tg mice, for different A β-shapes The dot blot assay of the antibody repertoire of formula

With the A β immunity inoculation APP/L mice of multi-form (Moechars etc., 1999, J.Biol.Chem.274, 6483-6492) after (reference example 4), evaluate plasma sample anti-A β antibody.To this end, be supplemented with 0.2mg/ml BSA's PBS is prepared for the scope serial dilution thing in single A β (1-42) form of 100pmol/ μ l-0.01pmol/ μ l.By 1 μ l's Every kind of sample is imprinted on nitrocellulose membrane.Corresponding mice plasma sample (1: 400 dilution) is employed in order to detect.Use is sewed Anti-mouse-the IgG and stain NBT/BCIP that close alkali phosphatase carry out immunostaining.

A β-standard sample for Dot blot:

1.A β (1-42) ball aggressiveness

A β (1-42) ball aggressiveness goods are described in embodiment 1a.

A β (1-42) monomer of 2.HFIP pretreatment, is dissolved in Pluronic F68

By 3mg A β (1-42) (Bachem Inc. in 1.7ml Eppendorff pipe；Catalog number H-1368) molten Solution (Eppendorff hot mixing device, the 1400rpm) 1.5 that vibrate in 0.5ml HFIP (6mg/ml suspension) and at 37 DEG C is little Shi Zhizhi obtains settled solution.Drying sample (1.5 hours) being resuspended in 13.2 μ l DMSO in SpeedVac concentrator, Vibrate 10 seconds, be subsequently ultrasonicated for (20 seconds), and vibrate (as in Eppendorff hot mixing device, 1400rpm) 10 minutes.Add Add 6ml 20mM NaH₂PO₄；140mM NaCl；0.1%Pluronic F68；PH 7.4 is also stirred at room temperature 1 hour.? Centrifuge A sample 20 minutes under 3000g.Abandoning supernatant and by resolution of precipitate at 0.6ml 20mM NaH₂PO₄；140mM NaCl； In 1%Pluronic F68, pH 7.4.Add 3.4ml H₂O is also stirred at room temperature 1 hour, is then centrifuged 20 under 3000g Minute.8 parts of aliquots (every part of 0.5ml supernatant) are housed under-20 ° for further.

3.A β (20-42) ball aggressiveness

A β (20-42) ball aggressiveness goods are described in embodiment 1c.

4.A β (12-42) ball aggressiveness

A β (12-42) ball aggressiveness goods are described in embodiment 1d.

A β (1-40) monomer of 5.HFIP pretreatment, 5mM is dissolved in DMSO

1mg A β (1-40) (Bachem Inc, catalog number H-1194) is suspended in by Eppendorff pipe In 0.25ml HFIP (4mg/ml suspension).Vibrate at 37 DEG C this pipe (as in Eppendorff hot mixing device, 1400rpm) 1.5 hours to obtain settled solution, is then dried (1.5 hours) in speed vac concentrator.By molten for sample weight In 46 μ l DMSO (21.7 mg/ml solution=5mM), vibrate 10 seconds and be subsequently ultrasonicated for 20 seconds.Vibrated at 10 minutes (as In Eppendorff hot mixing device, 1400rpm) after by Sample Storage at-20 DEG C for further.

6.A β (1-42) monomer, 0.1%NH₄OH

1mg A β (1-42) (Bachem Inc., catalog number H-1368) is dissolved in 0.5ml 0.1%NH₄OH water In solution (freshly prepared) (=2mg/ml) and the most at room temperature vibration 30 seconds to obtain settled solution.By Sample Storage For further at-20 DEG C.

7.A β (1-42) fibril

1mg A β (1-42) (Bachem Inc. catalog number H-1368) is dissolved in 500 μ l 0.1%NH₄OH is water-soluble In liquid (Eppendorff pipe) be stirred at room temperature sample 1 minute.With 300 μ l 20mM NaH₂PO₄；140mM NaCl, pH With this freshly prepared A β (1-42) solution of 100 μ l in 7.4.PH to pH 7.4 is adjusted with 1%HCl.Incubated samples 24 at 37 DEG C Hour and be centrifuged (under 10000g, 10 minutes).Abandoning supernatant and by vortex 1 minute with 400 μ l 20mM NaH₂PO₄； The resuspended fibril of 140mM NaCl, pH 7.4 precipitates.

8.sAPPα

Come from Sigma (catalog number S9564；25 μ g are dissolved in 20mM NaH₂PO₄；140mM NaCl；In pH 7.4). Use 20mM NaH₂PO₄, 140mM NaCl, pH 7.4,0.2mg/ml BSA dilution sAPP α to 0.1mg/ml (=1pmol/ μ l).

Material for Dot blot:

A β-standard sample:

It is dissolved in 20mM NaH₂PO₄, the serial dilution of the A beta antigen in 140mM NaCl, pH 7.4+0.2mg/ml BSA

1)100pmol/μl

2)10pmol/μl

3)1pmol/μl

4) 0,1pmol/ μ l

5) 0,01pmol/ μ l

Celluloid:

Electro-blotting (Trans-Blot) transfer medium, the nitrocellulose membrane (0.45 μm) of cleaning；BIO-RAD

Anti-mouse-AP:

AQ330A(Chemicon)

Detectable:

NBT/BCIP sheet (Roche)

Bovine serum albumin (BSA):

A-7888(SIGMA)

Closed reagent:

5% low fat milk TBS solution

Buffer solution:

TBS

25mM Tris/HCl-pH of buffer 7.5

+150mM NaCl

TTBS

25mM Tris/HCl-pH of buffer 7.5

+150mM NaCl

+ 0.05%Tween 20

PBS+0.2mg/ml BSA

20mM NaH₂PO₄PH of buffer 7.4

+140mM NaCl

+0.2mg/ml BSA

Antibody-solutions I:

The mice plasma sample of the active immunization research that A β (20-42) ball aggressiveness of using by oneself is carried out is (at 20ml 1% With 1: 400 dilution in low fat milk TBS solution)

Antibody-solutions II:

1: 5000 dilution

Anti-mouse-AP, is dissolved in 1% low fat milk TBS solution

Dot blot step:

1) every kind of different A β-standard sample of 1 μ l (in their 5 times of serial dilutions) is imprinted on nitrocellulose membrane, About 1cm apart.

2) under room temperature (RT), (=speckle prints to air-dry the A β-standard sample speckle at least 10 minutes on nitrocellulose membrane Mark)

3) close:

30ml 5% low fat milk TBS solution incubation Dot blot is used 1.5 hours under RT.

4) washing:

Discard lock solution and use 20ml TTBS incubated under agitation Dot blot 10 minutes under RT.

5) antibody-solutions I:

Discard lavation buffer solution and use antibody-solutions I incubation Dot blot overnight under RT.

6) washing:

Discard antibody-solutions I and use 20ml TTBS incubated under agitation Dot blot 10 minutes under RT.Discard wash solution also 20ml TTBS incubated under agitation Dot blot is used 10 minutes under RT.Discard wash solution and by 20ml TBS vibration temperature under RT Educate Dot blot 10 minutes.

7) antibody-solutions II:

Discard lavation buffer solution and use antibody-solutions II incubation Dot blot 1 hour under RT.

8) washing:

Discard antibody-solutions II and use 20ml TTBS incubated under agitation Dot blot 10 minutes under RT.Discard wash solution And use 20ml TTBS incubated under agitation Dot blot 10 minutes under RT.Discard wash solution and vibrate with 20ml TBS under RT Incubation Dot blot 10 minutes.

9) development:

Discard wash solution.1 NBT/BCIP is dissolved in 20ml H₂In O and with this solution incubation Dot blot 5 points Clock.By with H₂O thoroughly washs termination development.

Result is shown in Figure 4.

Dot blot point is carried out to anti-A β antibody produced after A β (20-42) ball aggressiveness active immunization mice Analysis is to evaluate they specificitys to the A β of multi-form.The A β of single form is imprinted on film with serial dilution form and with contain There is the corresponding mice plasma incubation of the anti-A β-antibody produced in immunoreaction process.Single Dot blot is corresponding to difference Immune mouse individual.

1.A β (1-42) ball aggressiveness

2.A β (1-42) monomer, HFIP pretreatment, it is dissolved in 0.1%Pluronic F68

3.A β (20-42) ball aggressiveness

4.A β (12-42) ball aggressiveness

5.A β (1-40) monomer, HFIP pretreatment, 5mM DMSO solution

6.A β (1-42) monomer, 0.1%NH₄OH

7.A β (1-42) fibril goods

8.sAPPα(Sigma)；(the first speckle: 1pmol)

In the active immunization of APP/L Tg-mice is studied, show that compared with processing with PBS, these are little alleviating Mus cognitive defect creates best result by A β (20-42) ball aggressiveness immunity inoculation.A β (20-42) ball aggressiveness of using by oneself is certainly The plasma sample of the APP/L Tg mice after dynamic immunity inoculation has and is similar to the most claimed A β (20-42) ball and gathers The antibody repertoire (main identification A β (20-42) ball aggressiveness and A β (12-42) ball aggressiveness) of body mAbs.

Embodiment 6: with A β (1-42) monomer, A β (1-42) ball aggressiveness, A β (20-42) ball aggressiveness or the carrier as comparison Solubility and insoluble A β (1-42) and A β (1-40) peptide in the brain extract of the APP/PS1 Tg mice of active immunization Concentration

For this research, employ the female mice (Alzheimer in FVBxC57B1/6J background at 40 4 monthly ages Sick double transgenic mouse model (APP/PS1 Tg mice)).APP/PS1 Tg mice contains from V717I, (position refers to It is the APP-isotype grown most) 695 aminoacid of people APP of suddenling change and the other people's aging egg with A264E sudden change White 1 gene.Under the two gene controls all in Thy1 promoter.At reMYND, the Experimental Genetics In one Basic Laboratory of Group, Campus Gasthuisberg, Catholic University Leuven, by Fred The breeding such as Van professor Leuven also identifies mice.

All 3 week old mices are carried out gene type by polymerase chain reaction (PCR), once knows PCR result, just give Give unique identification number.

The Mus food of mice energy contact free pre-filtering and aseptic water (UV-lamp) and standard.By food under Dry and cold circumstance It is housed in draughty storeroom.Water of daily check and the amount of food, supplement when needing, and sets and updates weekly two Secondary.

Under anti-circadian rhythm: 14 hours illumination/10 hour started at 7 in afternoon are dark, allow mice stay in the RVS of standard T2 type metal cage (area 540cm²In).Cage is equipped with hard floor and the Radix Glycyrrhizae layer as bedding.According to Animal Welfare Law The mice quantity of restrictions system every cage.Before performance testing starts 5 days, mice is transferred to macrolon 2 type cage and transports It is passed to laboratory, in order to adapt to laboratory environment and prepare performance testing.

The 100 μ g that in mouse peritoneum, acceptance mixes with equivalent complete Freund's adjuvant are dissolved in phosphate buffered saline (PBS) (PBS) In A β (1-42) monomer (0.1%NH₄OH), A β (1-42) ball aggressiveness or A β (20-42) ball aggressiveness, then every three weeks use Same amount of antigen booster injection in incomplete Freund's adjuvant, continue 4 months.

Biochemistry

The A β (1-40) in 1 hemisphere soluble fraction and A β (1-42) is measured by ELISA.Additionally, surveyed by ELISA The A β (1-40) and A β (1-42) of fixed 1 hemisphere insoluble film fraction.

With salt ketamine (Ketalar) (ketamine), xylazine (Rompun) (2% xylazine) and atropinic 2: 1: 1 mixture anesthetized mice also rinses through cardia physiological serum at 4 DEG C.Carry out this flushing to remove blood from cerebrovascular Liquid, this operation does not affect organ integrity.

In neck muscle between skull and atlas bone, otch collects cerebrospinal fluid (CSF).Use No. 26 metered shot Pin carries out cisternal puncture and collects 10-20 μ l CSF with thin glass pipette.

By cardiac puncture and 1ml syringe by blood collecting in heparin coated Eppendorf pipe.At 4 DEG C with 14.000rpm centrifugal blood 5 minutes.At serum is housed in-70 DEG C.

Mice is rinsed through cardia physiological serum at 4 DEG C.

Brain is separated with skull, and separates hindbrain and forebrain by coronalplane/face amount cutting.Discard cerebellum.By using Forebrain is bisected into left hemisphere and right hemisphere by the cutting of center line sagittal.

One hemisphere is immersed in liquid nitrogen immediately and is housed at-70 DEG C until biochemical analysis.

The homogenate of one brain hemisphere and fractionated

Use Potter, glass tubing (without detergent, 2cm³) and mechanical homogeniser (650rpm) carry out brain homogenate.6,5 x 1/2 brain weight volume freshly prepared containing protease inhibitor (every 50ml Tris/HCl buffer 1, Complete^TM, Roche, Mannheim, Germany) 20mM Tris/HCl buffer (pH 8.5) be used as homogenate buffer.

Sample is transferred to from-70 DEG C in the shuttle of band liquid nitrogen, and by incubation on the table before homogenate Within several seconds, preheat each sample.Homogenate is collected in Beckman centrifuge tube TLX and before centrifugation in cooled on ice.At two kinds Between sample, with the carefully rinsing Potter of the distilled water (AD) without detergent and glass tubing and it is dried with absorbent paper.

At 4 DEG C with 48000rpm (135.000xg) pre-cooling supercentrifuge (Beckman, Mannheim, Germany) Centrifuge A sample 1 hour 20 minutes in.Due to the centrifuge support (N=8) of limited quantity, classified by brain weight Sample (with equilibrium centrifugation machine) is also upset at random, in order to be divided into different process groups on different centrifugation times.

By supernatant (APP containing secretion and the soluble fraction of amyloid peptide) with precipitation (at aged mice In the case of, the film fraction of the APP-fragment amyloid peptide relevant with speckle is combined containing film) separate.Supernatant is divided into two Pipe, wherein a pipe is standby at being housed in-20 DEG C, and another pipe carries out further column chromatography process to concentrate amyloid peptide.

Brain weight, the Tris/HCl buffer volume of use, centrifugation time (passing through color mark) and for column chromatography Soluble fraction partial volume is given in following table illustratively.

Little reversed-phase column (C18-Sep-Pack Vac 3cc cylinder, Waters, Massachusetts, MA) is arranged on In vacuum system and with 80% acetonitrile (A-TFA) washing being dissolved in 0.1% trifluoroacetic acid, then wash two with 0.1%TFA Secondary.Then apply a sample to wash on pillar and with successively 5% and 25%A-TFA.With 75%A-TFA eluting amyloid Eluent is also collected in 2ml pipe by peptide on ice.Freezing in SpeedVac concentrator (Savant, Farmingdale, NY) Dry eluent overnight, is laid equal stress on and is dissolved in the diluents that 330 μ l ELISA kit provide.

Precipitation is classified into different film fraction further: film fraction A (MFA), film fraction B (MFB) containing total length APP and Film fraction C (MFC) containing the relevant amyloid of speckle.Therefore, will be precipitated and dissolved in containing protease inhibitor (every 50ml TBS Buffer 1, Complete^TM, Roche, Mannheim, Germany) TBS buffer in, and MFA is divided into two pipes, wherein One pipe is standby at being housed in-20 DEG C.Add the NP40 (2% final volume) and Triton being dissolved in the TBS containing protease inhibitor X-100 (2% final volume) processes further the MFA of 60%, and uses level (swing-out) rotor (SW60), at 4 DEG C in Beckman supercentrifuge is centrifuged 1 hour with 27.000rpm (98 ' 000xg).By supernatant (MFB) and precipitation (MFC) phase At separating and being all housed in-20 DEG C.

Brain weight, the brain weight of 60%, the TBS+PI+NP40+Triton X-100 buffer volume of use and centrifugal Time (passing through color mark) is given in following table illustratively.

The ELISA of the people A β in the soluble fraction of a hemisphere

For people A β (1-40) and the amount of people A β (1-42) in the soluble fraction of quantitative brain homogenate and/or cerebrospinal fluid (CSF), Use commercially available enzyme-linked immunosorbent assay (ELISA) test kit (ELISA is highly sensitive for people's amyloid-beta 40 or β 42, The Genetics Company, Zurich, Switzerland).ELISA is carried out according to manufacturer's experimental program.In short, There is no system in the 96-hole Polypropylene plates (Greiner bio-one, Frickenhausen, Germany) of protein binding capacity Standby standard sample (the A β (1-40) of synthesis or the dilution of A β (1-42)) and sample.The sample provided in ELISA kit is dilute Release in agent preparation and have 1000,500,250,125,62.5,31.3 and the standard sample dilution of 15.6pg/ml final concentration and sample Product are to the final volume of 60 μ l.Owing to amyloid levels increases with Mouse Age, and actual evaluation needs sample readout It is in the linear segment of standard curve, thus be accordingly used in sample 1: 3 dilution that A β (1-40) analyzes, analyze for A β (1-42) Sample 1: 6 dilutes.

Sample, standard sample and blank (50 μ l) are added to and (catches and select in anti-A β-coated polystyrene plate The antibody of property identification antigens c-end), this flat board also carries selective anti-A β-antibody conjugates (biotinylated detection in addition Antibody), and be incubated overnight at 4 DEG C, in order to antibody-amyloid-antibody complex is formed.Second day, add chain Mould Avidin-Peroxidase-conjugate, adds TMB/ peroxide mixture, makes substrate be changed into after 30 minutes Color products.Stop this reaction by adding sulphuric acid (1M) and carry out luminosity survey by the microplate reader utilizing band 450nm optical filter Determine thus measure color intensity.Absorbance is compared by the standard curve prepared with the A β (1-40) or A β (1-42) that use synthesis Obtain the quantitative of sample A β content.

The ELISA of the people A β in the insoluble fraction of a hemisphere

For people A β (1-40) in the insoluble film fraction of quantitative brain homogenate and the amount of people A β (1-42), MFC sample is carried out Process further and be dissolved in 8M guanidine 80mM Tris/HCl solution.Then at 25 DEG C in hot mixing device (thermomixer) Incubated samples 3 hours and per hour with the 100 μ reciprocal imbibitions of l pipet so that MFC resolution of precipitate is entered in guanidine buffer.Finally, exist Under 4000rpm, Centrifuge A sample only 1 minute is to remove chip.

Brain weight, MFC Sediment weight and 8M guanidine buffer volume are given in following table illustratively.

For people A β (1-40) in quantitative final sample and the amount of people A β (1-42), employ commercially available enzyme-linked immunosorbent assay (ELISA) test kit (42ELISA is highly sensitive for people's amyloid-beta 40 or β, The Genetics Company, Zurich, Switzerland).In addition to prepared by standard sample (dilution of A β (1-40) or A β (1-42) of synthesis), root ELISA is carried out according to manufacturer's experimental program.Sample is prepared to the whole body of 60 μ l in the diluents that ELISA kit provides Long-pending.Owing to guanidine have impact on the OD-value of standard curve, therefore prepare in there is the diluents of guanidine of same concentrations with sample Final concentration 1000,500,250,125,62.5,31.3 and the standard dilutions of 15.6pg/ml.It is not having protein binding capacity 96-hole Polypropylene plates (Greiner bio-one, Frickenhausen, Germany) in carry out.

Owing to amyloid levels increases with Mouse Age, and actual evaluation needs sample readout to be in standard song In the linear segment of line, thus be accordingly used in insoluble A β (1-40) and sample 1: 500 dilution that insoluble A β (1-42) analyzes.

Result is shown in Figure 5.

With A β (1-42) monomer (0.1%NH₄OH), A β (1-42) ball aggressiveness, A β (20-42) ball aggressiveness or as comparison Solubility and insoluble A β (1-42) and A β (1-in the brain extract of the APP/PS1 Tg-mice of carrier active immunization 40) concentration of peptide.

By the solubility of the APP/PS1 Tg-mouse brain extract of A β (20-42) ball aggressiveness active immunization and not In soluble fraction, the level of A β (1-40)-and A β (1-42)-peptide is not significantly different from vehicle Control.In contrast, A β is used (1-42) ball aggressiveness and A β (1-42) monomer immunity inoculation cause brain A β (1-40)-and the subtracting of A β (1-42) level.This shows A β (20-42) ball aggressiveness direct immunization inoculation method will not significantly change total A β-brain level, however still can be effectively Alleviate the cognitive defect (seeing embodiment 4) that A beta-peptide is relevant.

Embodiment 7: with after anti-A β (20-42) ball dimer antibody passive immunization, by object identification test analysis APP/L transgenic mice cognitive performance

In these experiments, the process LAN mice with the people APP of point mutation is employed.This point mutation refers to aminoacid 717 (isoleucine instead of valine) has also seen in London (London) family, and in this family, it caused AD to send out before 60 years old Make (Mullan etc., Nature Genetics 2 (1992) 340-342).Referred to here as the transgenic mice of APP/L is Leuven Created and described (Moechars etc., J.Biol.Chem.274 (1999) 6483-6492) first.Female APP/ to 3 monthly ages L mice carries out passive immunization.Mice accepts any one of the 250 μ g being dissolved in 100 μ l phosphate buffered saline (PBS) (PBS) Monoclonal mouse antibody 5F7,10F11 or 7C6.In whole experimentation, (start from afternoon 7 in contrary day night circulation 14 hours illumination/10 hour of point are dark) in animal is remained at standard under conditions of.They have tolerated passively well Immunity inoculation, does not has the symptom of any adverse effect.

After third time injection (the 15th day of experiment), tested by object identification the most described in the art The cognitive competence of the inspection such as (Dewachter Journal of Neuroscience 22 (2002) 3445-3453) mice.For This, allow mice get used to playground and to be then exposed to detect 10 minutes (acquisition) stages, during this period by them It is individually placed in the playground now containing two similar elements (green cubic body similarly sized for about 4cm or orange cylinder) In.The persistent period of record mice detecting object and frequency.During memory stage, after 2.5 hours, mice is sent back to except Now contain outside knowing object in the playground of other objects.To the identification record of new object for (to detect old relative to total time With new object) mice detection past heritage body during time." discrimination index (recognition index) " have expressed this The relation of kind (for the time/total time of new object).The mice not remembeing known object is considered as that it is same to new object Interested and spending same time detects, and in other words, has the discrimination index of 50%.Remember that the mice of known object is recognized For being different interested and therefore there is considerably higher discrimination index.Known APP/L mice is cognitive at 4.5 monthly ages Defect, and there is the discrimination index in Random Level (i.e. 50%) yardstick.

Result is shown in Figure 6.

Object identification test is carried out in mice.This test report is according to the detection row during 10 minutes test phases By the identification with unknown object phase comparison known object measured.Discrimination index is defined relative to spend in two kinds of things of detection Time on body, mice spent percentage of time on detection unknown object.At test phase first 2.5 hours, at 10 minutes During detection phase, known object is exposed to mice.

A) exempted from by peritoneal injection 250 μ g antibody 5F7 (n=9), antibody 10F11 (n=11) or antibody 7C6 (n=11) Epidemic disease APP transgenic mice, once in a week, continues three weeks；Control animal accepts PBS (n=6).With Random Level (50%, the most colored The time taken on the known and unknown object of detection is equal) significant difference is expressed as asterisk.*=p ＜ 0.05 (t-inspection)

B) useful antibody (5F7,10F11 and 7C6 are compared；(n=31) mice that) processes and use phosphate buffered saline (PBS) (PBS；N=6) mice processed.The RI of antibody treatment group and Random Level significant difference (* *=P ＜ 0.01；T-checks).

Known APP/L mice is cognitive defect at 4.5 monthly ages, and has the knowledge in Random Level (i.e. 50%) yardstick Other index.

It practice, the mice that PBS-processes shows random behavior.With all three antibody (5F7,10F11 and 7C6) quilt Dynamic immunity inoculation result in the discrimination index dramatically increased.When as mixing group compared with the control, discrimination index dramatically increases. After giving all three antibody, this beneficial effect of the memory performance of APP/L mice is implied the A β (20-42) for truncate The antibody of ball aggressiveness be enough to realize cognitive improvement.

Embodiment 8: anti-A β (20-42) the selective Dot blot of ball dimer antibody is composed

In order to characterize the selectivity of monoclonal anti A β (20-42) ball dimer antibody, detect they and different A β-form Identify.To this end, single at 100pmol/ μ l-0.01pmol/ μ l of the scope that is prepared in the PBS being supplemented with 0.2mg/ml BSA The serial dilution of A β (1-42) form.Every kind of sample of 1 μ l is imprinted on nitrocellulose membrane.Corresponding resisting is employed in order to detect Body (0.2 μ g/ml).Use and put together the anti-mouse-IgG of peroxidase and stain BM Blue POD substrate (Roche) enters Row immunostaining.

A β-standard sample for Dot blot:

1.A β (1-42) monomer, 0.1%NH₄OH

2.A β (1-40) monomer, 0.1%NH₄OH

1mg A β (1-40) (Bachem Inc., catalog number H-1368) is dissolved in 0.5ml 0.1%NH₄OH water In solution (freshly prepared) (=2mg/ml) and the most at room temperature vibration 30 seconds to obtain settled solution.By Sample Storage For further at-20 DEG C.

3.A β (1-42) monomer, 0.1%NaOH

2.5mg A β (1-42) (Bachem Inc., catalog number H-1368) is dissolved in 0.5ml 0.1%NaOH water In solution (freshly prepd) (=5mg/ml) and the most at room temperature vibration 30 seconds to obtain settled solution.By Sample Storage- For further at 20 DEG C.

4.A β (1-40) monomer, 0.1%NaOH

2.5mg A β (1-40) (Bachem Inc., catalog number H-1368) is dissolved in 0.5ml 0.1%NaOH water In solution (freshly prepd) (=5mg/ml) and the most at room temperature vibration 30 seconds to obtain settled solution.By Sample Storage- For further at 20 DEG C.

5.A β (1-42) ball aggressiveness

A β (1-42) ball aggressiveness goods are described in embodiment 1a.

6.A β (12-42) ball aggressiveness

A β (12-42) ball aggressiveness goods are described in embodiment 1d.

7.A β (20-42) ball aggressiveness

A β (20-42) ball aggressiveness goods are described in embodiment 1c.

8.A β (1-42) fibril

9.sAPPα

Material for Dot blot:

A β-standard sample:

1)100pmol/μl

2)10pmol/μl

3)1pmol/μl

4) 0,1pmol/ μ l

5) 0,01pmol/ μ l

Celluloid:

Anti-mouse-POD:

Catalog number: 715-035-150 (Jackson Immuno Research)

Detectable:

BM Blue POD substrate, (Roche) of precipitation

Bovine serum albumin, (BSA):

Catalog number: A-7888 (SIGMA)

Closed reagent:

5% low fat milk TBS solution

Buffer solution:

TBS

25mM Tris/HCl pH of buffer 7.5

+150mM NaCl

TTBS

25mM Tris/HCl-pH of buffer 7.5

+150mM NaCl

+ 0.05%Tween 20

PBS+0.2mg/ml BSA

20mM NaH₂PO₄PH of buffer 7.4

+140mM NaCl

+0.2mg/ml BSA

Antibody-solutions I:

0.2 μ g/ml antibody of dilution in 20ml 1% low fat milk TBS solution

Antibody-solutions II:

1: 5000 dilution

Anti-mouse-POD, is dissolved in 1% low fat milk TBS solution

Dot blot step:

3) close:

4) washing:

5) antibody-solutions I:

Discard lavation buffer solution and use antibody-solutions I incubation Dot blot 2 hours under RT.

6) washing:

7) antibody-solutions II:

Discard lavation buffer solution and use antibody-solutions II incubation Dot blot overnight under RT.

8) washing:

9) development:

Discard wash solution.With 10ml BM Blue POD Substrate development Dot blot 10 minutes.By with H₂O is thorough Washing Dot blot terminates development.Use photodensitometry (GS800 photodensitometer (BioRad) and software kit Quantity One, 4.5.0 version (BioRad)) carry out the quantitative assessment of spot intensity.Only have relatively at the final optically clear A β identified (20-42) speckle of the relative density of relatively denser the 20% of ball aggressiveness speckle is evaluated.Independent to each Dot blot Measure this threshold value.The instruction of this value of calculation is identifying between A β (20-42) ball aggressiveness and corresponding A beta form for given antibody Relation.

Result is shown in Figure 7.

To different anti-A β antibody (6E10,5F7,4B7,10F11,6A2,4D10,2F2；3B10、7C6、7E5、10C1) Specificity for the A β of multi-form carries out dot blot assay.By little with the aggressiveness active immunization of A β (20-42) ball Mus, the hybridoma then merged by selection, it is thus achieved that the monoclonal antibody (in addition to 6E10) tested.By single A beta form Serial dilution is also used for immunoreation with corresponding monoclonal antibody incubation.

1.A β (1-42) monomer, 0.1%NH₄OH

2.A β (1-40) monomer, 0.1%NH₄OH

3.A β (1-42) monomer, 0.1%NaOH

4.A β (1-40) monomer, 0.1%NaOH

5.A β (1-42) ball aggressiveness

6.A β (12-42) ball aggressiveness

7.A β (20-42) ball aggressiveness

8.A β (1-42) fibril goods

9.sAPPα(Sigma)；(the first speckle: 1pmol)

In distinguishing A β (1-42) ball aggressiveness and A β (12-42) ball aggressiveness, anti-A β (20-42) ball aggressiveness selectivity mAbs Can be divided three classes.The first kind includes antibody 6A2,5F7 and 2F2, and preferential identification A β (20-42) ball aggressiveness is also known to a certain extent Other A β (1-42) ball aggressiveness (and A β (12-42) ball aggressiveness).Equations of The Second Kind includes antibody 10F11,4D10 and 3B10, preferentially identifies A β (20-42) ball aggressiveness, also identifies A β (12-42) ball aggressiveness, but in lesser degree and and inconspicuous identification A β (1-42) ball Aggressiveness.3rd class includes antibody 7C6,4B7,7E5 and 10C1, identifies A β (20-42) ball aggressiveness, but display is not to other A β balls The obvious identification of aggressiveness.All three classes the most inconspicuous identification monomer A β (1-42), monomer A β (1-40), A β (1-42) fibril or sAPPα。

In passive immunization, the selectivity spectrum of anti-A β (20-42) ball dimer antibody shows significantly raised identification Index (in figure 6), due principally to A β (20-42) ball aggressiveness and A β (12-42) the ball aggressiveness of Selective recognition truncate, and Identification A β (1-42) the ball aggressiveness of not half and nonrecognition monomer A β (1-42), monomer A β (1-40), A β (1-42) fibril Dimension or sAPP α.

Embodiment 9:A β (20-42) antibodies selective and fibril shape A β peptide are (with A β plaque shape in old TG2576 mice That formula exists and exist with A beta amyloid protein form in meningovascular) in-situ study of specific reaction

For these test, employ 19 monthly ages TG2576 mice (Hsiao etc., 1996, Science；274 (5284), The APP/LxPS1 mice at 99-102) or 9 monthly ages (describes as above；ReMYND, Leuven, Belgium) brain material or two Ah Alzheimer's disease patient (RZ16 and RZ55；Be derived from BrainNet, Munich) autopsy material.Mice process LAN with So-called Swede sudden change (K670N/M671L；Tg2576) or so-called Londoner sudden change (V717I) people APP, additionally mistake Express with A264E sudden change people's presenilin 1 gene (APP/LxPS1), and when the about 7-11 monthly age in brain essence shape Become beta amyloid proteinosis thing, in bigger cerebral blood vessel, form beta amyloid proteinosis thing when about 18 monthly age (Tg2576).By animal deep anaesthesia and through perfusion of carotid artery 0.1M phosphate buffered saline (PBS) (PBS) to rinse blood.Then cranium Brain longitudinally spaced apart is taken off on bone.One hemisphere of quick freeze brain, another hemisphere is then by being immersed in 4% paraformaldehyde It is fixed.By sucking the fixing hemisphere of 30% sucrose PBS solution low-temperature protection submergence and being arranged on freezing microtome.Right Complete forebrain cuts 40 μm sections, is collected in PBS and for staining procedure subsequently.Human brain material is an about 1cm³The degree of depth The neopallium block freezed.A fraction of piece of submergence is fixed in 4% paraformaldehyde and enters as mouse brain material One step processes.

Use the following single section of experimental program congo red staining:

Material:

-amyloid dye Congo red test kit (Sigma-Aldrich；HT-60), by the NaCl solution containing ethanol, NaOH solution and Congo red solution composition

-dyeing cuvette

-microscope slide SuperfrostPlus and coverslip

-ethanol, dimethylbenzene, embedding medium

Reagent:

-produce base brine with NaCl solution 1: 100 dilution NaOH

-(load is made in using first 15 minutes to produce the red solution of alkaline Congo by Congo red solution 1: 100 dilution base brine Standby, filtrate)

-section is fixed on microscope slide and is allowed to dry

-dyeing cuvette in incubation microscope slide, first incubation 30-40 minute in base brine, then firm in alkalescence Incubation 30-40 minute in arnotto solution

-embed with fresh ethanol rinse 3 times and on dimethylbenzene

First with Zeiss Axioplan microscope to dyeing photograph qualitative evaluation.Red color instruction exists with speckle form And Amyloid deposits in bigger meningovascular.These results are shown in Fig. 8 A.After a while, evaluation concentrates on this The antibody staining of a little structures.

According to following experimental program, by section is resisted with the solution incubation containing 0.07-7.0 μ g/ml corresponding antibodies Body dyes:

Material:

-TBST cleaning mixture (the Tris buffer saline containing Tween 20；10x concentrates；DakoCytomation；S3306 1∶ 10 are dissolved in double distilled water)

-0.3%H₂O₂Methanol solution

-5% donkey serum (Serotec) TBST solution

-in TBST dilution Monoclonal mouse-anti-ball dimer antibody

-second antibody: biotinylated donkey-anti-mouse antibody (Jackson Immuno；715-065-150；With 1: 500 It is diluted in TBST)

-StreptAB complex (StreptABComplex) (DakoCytomation；K 0377)

-peroxidase substrate kit diaminobenzidine (=DAB；Vector Laboratories；SK-4100)

-SuperFrost Plus microscope slide and coverslip

-embedding medium (Medite without dimethylbenzene；X-tra Kitt)

Method:

-ice-cold 0.3%H is transferred in the section floated₂O₂In and incubation 30 minutes

-wash 5 minutes in TBST buffer

-and donkey serum/TBST incubation 20 minutes

-at room temperature with first antibody incubation 24 hours

-wash 5 minutes in TBST buffer

-with the closing Serum incubation 20 minutes from Vectastain Elite ABC peroxidase conjugation kit

-wash 5 minutes in TBST buffer

-at room temperature with second antibody incubation 60 minutes

-wash 5 minutes in TBST buffer

-at room temperature with StreptABComplex incubation 60 minutes

-wash 5 minutes in TBST buffer

-with the DAB incubation 20 minutes from Vectastain Elite ABC peroxidase conjugation kit

-section is fixed on microscope slide, air-dry, with ethanol dehydration and embed

In addition to visual inspection dyeing, by using ImagePro 5.0 image analysis system to cut from histology picture 10 specklees randomly choosed and measure their average gray value speckle dyeing is carried out extra quantitatively.By deducting from shallow lake The average background density of the coloring matter of powdered protein speckle density, according to gray value calculate optical density value (0%-without speckle dye, Above-mentioned environmental background, 100%-is without transmission/maximum dyeing), and examine respectively between comparison and antibody with ANOVA and 6G1 and selectivity are for the statistical significance of difference between the antibody of A β (20-42).

During in Tg2576 and APP/LxPS1 mice, coloration result is shown in Fig. 8 B-D and H.

Laterally cutting of the neopallium of the different antibodies under 0.7 μ g/ml concentration and transgenic TG 2576 mice at 19 monthly ages The combination of sheet:

C) essence A β deposit (amyloid speckle) only 6G1 and 6E10 is dyeed, and is not that ball aggressiveness selectivity resists Body (i.e. 5F7,2F2,6A2,4D10,10F11,3B10,7C6,7E5 and 10C1) is dyeed.

D) compared with commercial antibody 6E10 and 4G8, all ball aggressiveness antibodies selectives (i.e. 5F7,2F2,6A2,4D10, 10F11,3B10,7C6,7E5 and 10C1) all show that significantly less essence speckle dyes.

The neopallium of the transgenic APP/LxPS1 mice at different antibodies and 11 monthly ages under 0.07-7.0 μ g/ml concentration The combination of slices across:

E) with ball aggressiveness antibodies selective (i.e. 5F7,2F2,6A2,4D10,10F11,3B10,7C6,7E5 and 10C1) phase Ratio, essence A β deposit (amyloid speckle) is significantly more and is dyeed by 6G1,6E10 and 4G8 under lower concentration.

The most by demonstrating all of Amyloid deposits (Congo red addicted to congo red staining；See figure 8A).Band=100 μm.

Evaluation to brown DAB deposit shows A β-non-selectivity 6G1 and 6E10 antibody staining speckle and meningovascular, A β (20-42) ball aggressiveness antibodies selective 5F7,2F2,6A2,4D10,10F11,3B10,7C6,7E5 and 10C1 the most do not contaminate Color.This discovery confirms these antibody and A β fibril or the knot of other A beta substances being present in internal amyloid structures Close and do not exist or the most little.The combination of this minimizing is considered to decrease dissolves the soonest due to speckle and increases solvable raw A subsequently The side effect that β is caused or the danger of the neuroinflamation caused by speckle binding antibody and microglia interact.

Coloration result in people's Alzheimer brain is shown in Fig. 8 B, F-H.

The combination of the slices across of the neopallium of the different antibodies under 0.7 μ g/ml concentration and patient RZ55:

B) essence A β deposit (amyloid speckle) only 6G1 and 6E10 is dyeed, and is not that ball aggressiveness selectivity resists Body (i.e. 5F7,2F2,6A2,4D10,10F11,3B10,7C6,7E5 and 10C1) is dyeed.

F) compared with commercial antibody 6E10 and 4G8, all ball aggressiveness antibodies selectives (i.e. 5F7,2F2,6A2,4D10, 10F11,3B10,7C6,7E5 and 10C1) all show significantly less dyeing.

H) blood vessel A β deposit (arrow) be only 6G1 and 6E10 dyeed, and be not ball aggressiveness antibodies selective (i.e. 5F7, 2F2,6A2,4D10,10F11,3B10,7C6,7E5 and 10C1) dyeed.

G) with ball aggressiveness antibodies selective (i.e. 5F7,2F2,6A2,4D10,10F11,3B10,7C6,7E5 and 10C1) phase Ratio, essence A β deposit (amyloid speckle) is significantly more and is dyeed by 6G1,6E10 and 4G8 under lower concentration.

The most by demonstrating all of Amyloid deposits (Congo red addicted to congo red staining；See figure 8A)。

Evaluation to brown DAB deposit shows A β-non-selectivity 6G1 and 6E10 antibody staining speckle and meningovascular, A β (20-42) ball aggressiveness antibodies selective 5F7,2F2,6A2,4D10,10F11,3B10,7C6,7E5 and 10C1 the most do not contaminate Color.Compared with ball aggressiveness antibodies selective, commercial antibody 6E10 and 4G8 shows higher dyeing, but compared with 6G1 dyeing for weak.Should Discovery confirms the dyeing spectrum in APP transgenic mice, i.e. ball aggressiveness antibodies selective and A β fibril or other are present in body The combination of the A beta substance in interior amyloid structures does not exists or the most little.The combination to people amyloid of this minimizing It is considered to decrease and dissolves and increase subsequently side effect that solubility A β caused the soonest due to speckle or owing to speckle combines anti- The danger of the neuroinflamation caused by body and microglia interaction.

Embodiment 10: anti-A β-antibody titer and speckle in about one year TG2576 mice plasma after active immunization Trace selectivity is composed

In the last immunity inoculation of Tg 2576 mice (from embodiment 9) (with A β (20-42) ball aggressiveness, A β (12- 42) ball aggressiveness, A β (1-42) monomer and carrier) after about one year, evaluate anti-A β that plasma sample produced and yet suffered from anti- Body.To this end, be prepared for the concentration range multi-form at 100pmol/ μ 1-0.01pmol/ μ l in PBS+0.2mg/ml BSA The serial dilution thing of A β (1-42).Every kind of sample of 1 μ l is imprinted on nitrocellulose membrane.In order to detect employ the least Mus plasma sample (1: 400 dilution).Anti-mouse-the IgG and stain NBT/BCIP that put together alkali phosphatase is used to exempt from Epidemic disease dyes.Every kind of sample of 1 μ l is put on nitrocellulose membrane.Use suitable mice plasma sample (1: 400 dilution) Detect.Anti-mouse-the IgG and additional stain NBT/BCIP that put together alkali phosphatase is used to dye.

A β-standard sample for Dot blot:

1.A β (1-42) ball aggressiveness

The goods of A β (1-42) ball aggressiveness are described in embodiment 1a.

A β (1-42) monomer of 2.HFIP pretreatment, is dissolved in Pluronic F68

3.A β (20-42) ball aggressiveness

The goods of A β (20-42) ball aggressiveness are described in embodiment 1c.

4.A β (12-42) ball aggressiveness

The goods of A β (1-42) ball aggressiveness are described in embodiment 1d.

5.A β (1-40) monomer, HFIP pretreatment, 5mM is dissolved in DMSO

1mg A β (1-40) (Bachem Inc, catalog number H-1194) is suspended in by Eppendorff pipe In 0.25ml HFIP (4mg/ml suspension).Vibrate at 37 DEG C this pipe (as in Eppendorff hot mixing device, 1400rpm) 1.5 hours to obtain settled solution, is then dried (1.5 hours) in speed vac concentrator.By molten for sample weight In 46 μ l DMSO (21.7mg/ml solution=5mM), vibrate 10 seconds and be subsequently ultrasonicated for 20 seconds.Vibrated at 10 minutes (as In Eppendorff hot mixing device, 1400rpm) after by Sample Storage at-20 DEG C for further.

6.A β (1-42) monomer, 0.1%NH₄OH

7.A β (1-42) fibril

8.sAPPα

Material for Dot blot:

A β-standard sample:

1)100pmol/μl

2)10pmol/μl

3)1pmol/μl

4)0.1pmol/μl

5)0.01pmol/μl

Celluloid:

Anti-mouse-AP:

AQ330A(Chemicon)

Detectable:

NBT/BCIP sheet (Roche)

Bovine serum albumin, (BSA):

A-7888(Fa.SIGMA)

Closed reagent:

5% low fat milk TBS solution

Buffer solution:

TBS

25mM Tris/HCl-pH of buffer 7.5

+150mM NaCl

TTBS

25mM Tris/HCl-pH of buffer 7.5

+150mM NaCl

+ 0.05%Tween 20

PBS+0.2mg/ml BSA

20mM NaH₂PO₄PH of buffer 7.4

+140mM NaCl

+0.2mg/ml BSA

Antibody-solutions I:

With the blood plasma of TG2576 mice of the active immunization of 1/400 dilution in 20ml1% low fat milk TBS solution.

Antibody-solutions II:

1: 5000 dilution

Anti-mouse-AP, is dissolved in 1% low fat milk TBS solution

Dot blot step:

3) close:

4) washing:

5) antibody-solutions I:

6) washing:

7) antibody-solutions II:

8) washing:

9) development:

Result is shown in Figure 9.

For different antibody repertoires, for different A beta forms in Dot blot, the blood of the different immunity inoculation group of test Clear: a) A β (20-42) ball aggressiveness；B) A β (12-42) ball aggressiveness；C) A β (1-42) monomer, 0.1%NH₄OH；D) vehicle Control.

1.A β (1-42) ball aggressiveness

2.A β (1-42) monomer, HFIP pretreatment, it is dissolved in 0.1%Pluronic F68

3.A β (20-42) ball aggressiveness

4.A β (12-42) ball aggressiveness

5.A β (1-40) monomer, HFIP pretreatment, 5mM DMSO solution

6.A β (1-42) monomer, is dissolved in 0.1%NH₄In OH

7.A β (1-42) fibril goods

8.sAPPα(Sigma)；(the first speckle: 1pmol)

Contrast with using the active immunization as the carrier compareed or A β (1-42) monomer, gather with A β (20-42) ball Body or A β (12-42) are even if ball aggressiveness immunity inoculation immunity inoculation the last time still has high antibody effect after about one year Valency.In the case of A β (20-42) ball aggressiveness immunity inoculation, these antibody selectivitys are for A β (20-42) ball aggressiveness, or at A β (12-42), in the case of ball aggressiveness immunity inoculation, these antibody are A β (20-42) ball aggressiveness and the selection of A β (12-42) ball aggressiveness Property.This shows that A β (20-42) the ball aggressiveness of truncate and A β (12-42) ball aggressiveness are extraordinary antigen, and for them Antibody retain the most long in vivo.

(noticing and observe unspecific staining signal on some Dot blot, it is likely to murine antibody and at A β Caused by BSA cross reaction used in the serial dilution of peptide)

Embodiment 11: the brain level of A β (20-42) ball mer epitopes in patients with Alzheimer disease

SDS-DTT-brain extract:

AD brain sample: RZ 16；RZ 52 and RZ 55 (being derived from Brain Net, Munich)

Control sample: RZ 92 (being derived from Brain Net, Munich)

1 Complete protease inhibitor (Roche, catalog number 1,697 498) is dissolved in 1ml H₂In O (= Protease inhibitor solutions).In glass jar, tap 20 homogenizing be dissolved in 2.5ml NaH₂PO₄, 140mM NaCl, 0.05% 100mg AD brain sample in Tween 20,0.5%BSA (being supplemented with 25 μ l Protease inhibitor solutions).At the most ultrasonic place Reason suspension 30 seconds, then incubation 16 hours at 37 DEG C.At 100'000g and 8 DEG C, centrifuged suspension 1 hour, then collects Supernatant.Residue is dissolved in 5mM NaH₂PO₄, tap in 35mM NaCl, pH 7.4 and in glass jar and be allowed to equal 10 times Matter.Add 75 μ l 10%SDS and 125 μ l 0.16mg/ml DTT and be stirred at room temperature 20 minutes.It is centrifuged under 10 ' 000g Sample 10 minutes, and at supernatant is overnight housed in-20 DEG C.Before the use, defrosting supernatant and under 10 ' 000g again from The heart 10 minutes.By supernatant (=SDS/DTT brain extract) for ELISA.

A) for the sandwich-ELISA of A β (20-42) ball mer epitopes

Reagent list:

1.F96Cert.Maxisorp NUNC-immuno plate (Immuno Plate) (catalog number: 439454)

2. binding antibody: 5F7,7C6,10F11

3. coupling buffer:

100mM sodium bicarbonate, pH9.6

4.ELISA closed reagent (Roche Diagnostics GmbH catalog number: 1112589)

5.PBST buffer:

20mM NaH₂PO₄, 140mM NaCl, 0.05%Tween 20, pH 7.4

6.A β (20-42) calibration standard

7. first antibody:

Anti-A β pRAb BA199；(by A β (1-42) ball aggressiveness-agarose gel (Sepharose)) of affinity purification IgG PBS solution；Concentration: 0.22mg/ml

8. second antibody:

Anti-rabbit-POD conjugate；(Jackson ImmunoResearch, catalog number: 111-036-045)

9. development:

-TMB；(Roche Diagnostics GmbH catalog number: 92817060) 42mM DMSO solution

-3%H₂O₂Aqueous solution

-100mM sodium acetate, pH4.9

-stopping solution: 2M sulphuric acid

Prepared by reagent:

1. binding antibody:

In coupling buffer, single binding antibody 5F7,7C6 and 10F11 are diluted to the final concentration of 0.7 μ g/ml.

2. closed reagent:

In order to prepare closing liquid storage, closed reagent is dissolved in 100ml H₂Store in O and with the aliquot of each 10ml At ensconcing-20 DEG C.

Use 27ml H₂O dilution 3ml closes liquid storage for closing one piece of elisa plate.

3.A β (20-42) calibration standard (CS1)

A β (1-42) ball aggressiveness goods are described in embodiment 1a.

A β (20-42) ball glycoprotein polyprotein precursor concentration (6.81mg/ml) is determined after Bradford (BioRad).By 14.68 μ L A β (20-42) ball aggressiveness (6.81mg/ml) is diluted in 10ml 20mM NaH₂PO₄, 140mM NaCl, 0.05%Tween20, PH 7.4, in 0.5%BSA (=10 μ g/ml).10 μ l 10 μ g/ml solution are diluted in 10ml 20mM NaH further₂PO₄, 140mM NaCl, 0.05%Tween20, pH 7.4, in 0.5%BSA (=10ng/ml=CS1).

Calibration standard for A β (20-42):

SDS/DTT-brain extract:

SDS/DTT-brain extract=E#

(# represents 4 kinds of human brain sample (1) RZ 16；(2)RZ 52；(3)RZ 55；(4)RZ 92)

Extract sample	Extract the volume of sample	PBST+0.5%BSA	Dilution factor
				E#.1	1ml E#	0.0ml	Directly
E#.2	0.316ml E#.1	0.684ml	1∶3.16
				E#.3	0.316ml E#.1	0.684ml	1∶10
E#.4	0.316ml E#.1	0.684ml	1∶31.6

4. first antibody:

In PBST+0,5%BSA, anti-A β pRAb liquid storage is diluted to 0.05 μ g/ml.Use this antibody-solutions immediately.

5. second antibody:

Anti-rabbit-POD the conjugate of lyophilizing is dissolved in 0.5ml H₂Mix in O and with 500 μ l glycerol.Then by antibody At concentrate is housed in-20 DEG C with the aliquot of 100 μ l.

With 1: 10 ' 000 dilution concentrate in PBST buffer.Use this antibody-solutions immediately.

6.TMB solution:

By 20ml 100mM sodium acetate, pH 4.9 mixes with 200 μ l TMB solution and 29.5 μ l 3% hydrogen peroxide.Vertical I.e. use this solution.

ELISA-plate for A β (20-42):

Measure twice calibration standard (CS1.1-CS1.8) and 4 kinds of human brain sample (1) RZ 16；(2)RZ 52；(3)RZ 55；(4) the SDS/DTT-brain extract (=E1-E4 is in their 4 serial dilution E#.1-E#.4) of RZ 92:

1

2

3

4

5

6

7

8

9

10

11

12

A

CS1.1

E1.1

E3.1

B

CS1.2

E1.2

E3.2

C

CS1.3

E1.3

E3.3

D

CS1.4

E1.4

E3.4

E

CS1.5

E2.1

E4.1

F

CS1.6

E2.2

E4.2

G

CS1.7

E2.3

E4.3

H

CS1.8

E2.4

E4.4

Each in monoclonal antibody 5F7 combined, 7C6,10F11 is carried out this ELISA.

Step

1. 100 μ l mAb solution are added in every hole.Under+6 DEG C (refrigerator), incubation elisa plate is overnight.

2. decantation antibody-solutions with PBST buffer solution hole three times, each 250 μ l.

3. add 250 μ l/ hole lock solution.At room temperature incubation 2 hours.

4. decantation lock solution with PBST buffer solution hole three times, each 250 μ l.

5. add every kind of calibration standard and the SDS/DTT brain extract in 100 μ l/ holes.At room temperature Incubate plates 2 is little Time, then at 6 DEG C overnight.

6. decantation calibration standard and SDS/DTT brain extract solution with PBST buffer solution hole three times, each 250 μl。

7. add first antibody solution at room temperature incubation 1 hour in 200 μ l/ holes.

8. decantation first antibody solution with PBST buffer solution hole three times, each 250 μ l.

9. add second antibody solution at room temperature incubation 1 hour in 200 μ l/ holes.

10. decantation second antibody solution with PBST buffer solution hole three times, each 250 μ l.

The 11. TMB solution adding 100 μ l/ holes.

12. during developing (at room temperature, 5-15 minute) monitoring board color, when applicable color development, by adding The stopping solution adding 50 μ l/ holes terminates reaction.

13. measure trap at 450nm.

14. use calibration calculations result.

15. evaluate: if the trap of sample is away from linear gauging scope, again dilutes them and repeat.

Result is shown in Figure 10.

The brain level of A β (20-42) ball mer epitopes in the brain extract that AD patient and comparison are individual

Sandwich ELISA is for evaluating A β (20-42) the ball mer epitopes content of brain extract truncate.Use for A β (20- 42) ELISA of the corresponding antibodies of ball aggressiveness is used as calibration.

Compared with comparison patient, the extract of Alzheimer brain tissue shows significantly raised A β (20- 42) ball mer epitopes content.This shows in fact A β (20-42) ball mer epitopes not only Ahl tribulus sea silent sickness animal model phase Close, or A substantia metachromatica granularis relevant in people's Alzheimer brain.Therefore, for the antibody of A β (20-42) ball mer epitopes It is to be expected to be useful in very much treatment Alzheimer.

Embodiment 12: develop anti-A β (20-42) ball aggressiveness hybridoma cell line

Multiple techniques known in the art can be used to prepare monoclonal antibody, including using hybridoma, recombinant and phagocytosis Body display technology or combinations thereof.Such as, hybridoma technology can be used to produce monoclonal antibody, described hybridoma technology includes Those are known in the art and such as at Harlow etc., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, second edition .1988)；Hammerling, etc., in:Monoclonal Antibodies (described list of references is with it at this most also for and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) Enter as reference) the middle technology instructed.As used herein, term " monoclonal antibody " is not limited by hybridoma technology The antibody produced.Term " monoclonal antibody " refers to that one derives from and includes any eukaryote, prokaryote or phage clone At interior monospecific polyclonal rather than derive from the antibody of method producing it.

As follows for producing the specific experiment scheme of antibody described herein:

The immunity inoculation of mice: the antigen subcutaneous immunizations Balb/c being dissolved in CFA with 50ug and A/J mice (6-8 week Age).Within every three weeks, it is dissolved in Immuneasy with 50ug^TM(Qiagen) antigen in is strengthened, and altogether strengthens three times.In fusion first 4 days, Mice is strengthened with the antigen intravenous of 10ug.

Cell merges and hybridoma screens: use standard technique to hang oneself in the future the splenocyte of immune animal and SP2/0-Ag14 Myeloma cell is merged with the ratio of 5: 1.After merging 7-10 days, when observing macroscopic colony, for for A β (20-42) antibody of ball aggressiveness checks SN by ELISA.Expanded by limiting dilution and clone from ELISA positive hole is thin Born of the same parents.

Antibody isotype measures: use Zymed EIA isotype kit measurement anti-A β (20-42) ball aggressiveness mAb's is same The type of kind.

The amplification of monoclonal antibody and purification: expand in the culture medium containing 5% low IgG hyclone (Hyclone) Hybridoma.Results supernatant also concentrates.Use purification mAb and dialyse in PBS.

Serum titer: with A β (20-42) ball 10 mices of aggressiveness immunity inoculation.The mice of all seroconversion all has 1: The ELISA titre (1/2 Max OD 450nm) of 5000-10,000.

********

Produce the name of the hybridoma of monoclonal antibody

Alpert laboratory internal for preserved material is named.

The cell line of preservation:

1) ML13-7C6.1D4.4A9.5G8 (referred to herein as " 7C6 ")

2) ML15-5F7.5B10 (referred to herein as " 5F7 ")

3) ML15-10F11.3D9 (referred to herein as " 10F11 ")

4) ML15-4B7.3A6 (referred to herein as " 4B7 ")

5) ML15-2F2.3E12 (referred to herein as " 2F2 ")

6) ML15-6A2.4B10 (referred to herein as " 6A2 ")

7) ML13-4D10.3F3 (referred to herein as " 4D10 ")

8) ML15-7E5.5E12 (referred to herein as " 7E5 ")

9) ML15-10C1.5C6.3H4 (referred to herein as " 10C1 ")

10) ML15-3B10.2D5.3F1 (referred to herein as " 3B10 ")

Claims

1. comprise Weight variable (VH) chain and the antibody of (VL) chain that can lighten, wherein said variable heavy chain and light chain CDR domain bag Aminoacid sequence containing selected from following CDR group:

2. the antibody of claim 1, wherein said antibody is humanized.

3. the antibody of claim 1 or 2, wherein said antibody comprises human constant region.

4. the antibody of any one of claim 1-3, wherein said antibody has people's glycosylation type.

5. the antigen-binding portion thereof of antibody as defined in any one of claim 1-4.

6. the antigen-binding portion thereof of claim 5, wherein said part is the Fab fragment of antibody, F (ab ')₂Fragment or scFv Fragment.

7. the nucleic acid of the separation of the antibody amino acids sequence of coding any one of claim 1-6.

8. comprise the carrier of the nucleic acid of the separation of claim 7.

9. comprise the host cell of the carrier of claim 8.

10. the method producing the antibody of any one of claim 1-4, described method is included in the condition being suitable for producing antibody Under, cultivate the host cell of claim 9 in the medium.

11. comprise as defined in any one of claim 1-6 antibody or antigen-binding portion thereof and pharmaceutically acceptable carrier Pharmaceutical composition.

12. antibody or antigen-binding portion thereof as defined in any one of claim 1-6 or are examined for treatment or prevention in preparation Application in the pharmaceutical composition of the amyloidosis of disconnected such as Alzheimer or mongolism.

The method of 13. amyloidosis diagnosing such as Alzheimer or mongolism, described method comprises offer From suspecting the individual sample suffering from amyloidosis, this sample is anti-with as defined in any one of claim 1-6 Body or antigen binding portion thereof, and detect the formation comprising antibody or antigen-binding portion thereof with the complex of antigen, exist multiple Compound then indicates individuality to suffer from amyloidosis.