HK1150061B

HK1150061B - Antibody and use thereof

Info

Publication number: HK1150061B
Application number: HK11104163.0A
Authority: HK
Inventors: 星美奈子; 佐藤道夫; 井手野祥次; 内藤幸嗣; 保理江智; 野田宗宏; 堀井肇
Original assignee: 道健康生活医药株式会社
Priority date: 2007-10-29
Filing date: 2008-10-29
Publication date: 2015-02-06

Description

Antibodies and uses thereof

Technical Field

The present invention relates to a novel antibody having low reactivity to amyloid precursor protein and high reactivity to amyloid spheres, and an application method thereof.

Background

Among various neurodegenerative diseases that develop with age, such as alzheimer's disease, parkinson's disease, huntington's chorea, and prion diseases, "abnormal structural proteins" have been attracting attention as a common pathogenesis, and research on the molecular nature thereof has been conducted. With respect to alzheimer's disease, the pathological features are reported to be the deposition in the brain of the following 2 fibrous aggregates: senile plaques containing amyloid beta (Abeta) as a main component (see Selkoe, D.J., Annu. Rev. Neurosci., 12, 463-890 (1989); and Glenner, G.G.and Wong, C.W., biochem. Biophys. Res. Commun., 120(3), 885-890(1984)) andneurofibrillary changes (paired helical fibers, PHF) with phosphorylated tau protein as the major component (Ihara, Y et al, J.biochem., 99, 1807-. In recent years, in the study of alzheimer's disease, which is considered to be caused by many and various causes, it has been considered that β amyloid aggregation is a common pathogenic pathway. Amyloid beta Protein is mainly 40 residues (a β) from its precursor substance (amyloid precursor Protein, APP)_1-40) 42 residues (A.beta.)_1-42) The peptide produced by cleavage as molecular species (molecules) is usually produced and decomposed in a monomeric form in healthy people, but in alzheimer's disease, aggregation of amyloid β is observed and excessive deposition is finally observed. The reason for this is considered to be that the control is out of control in the cutting-out process or the decomposing process. In the present specification, the former (A β) may be used_1-40) Referred to as "beta 40 amyloid", "beta 40 amyloid monomer" or "beta 40 amyloid monomer protein", and converts the latter (A beta)_1-42) Referred to as "beta 42 amyloid", "beta 42 amyloid monomer" or "beta 42 amyloid monomer protein". In addition, 43 residues (A.beta.. beta.)_1-43) The molecular species of (4) is produced by being cleaved even in a trace amount, and is sometimes referred to as "β 43 amyloid protein", "β 43 amyloid monomer" or "β 43 amyloid monomer protein".

Aggregated amyloid beta is thought to have a neurotoxic effect on nerve cells, causing synaptic degeneration and consequent nerve cell death, and is the mechanism of neuronal cell shedding leading to progressive cognitive impairment in alzheimer's disease. It is also reported that amyloid β does not show a neuronal death activity in a state released extracellularly as a water-soluble monomeric peptide (hereinafter, in the present specification, the neuronal death activity is sometimes referred to as "toxicity"), whereas toxicity begins to be obtained after the formation of amyloid β fibrils by self-association (refer to Lorenzo, a.and Yankner, b.a., proc.natl.acad.sci.usa., 91, 12243-containing 12247 (1994)). Since it is known that a toxic amyloid-beta-containing solution containing the amyloid-beta fibers causes death of cultured cells in the nervous system when added to these cells at a high concentration, it has been considered that the amyloid-beta fibers are a real cause of induction of death of nerve cells in alzheimer's disease.

Therefore, it is considered that an experimental system for inducing cell death of a nervous system cell or the like by adding a toxic β -amyloid containing the β -amyloid fiber reflects the nerve cell death of alzheimer's disease, and has been frequently used for screening of a nerve cell death inhibitor or the like. In recent years, however, the following facts have been gradually reported: (1) in a toxic amyloid β -containing solution containing amyloid β fibers, the concentration required for inducing neuronal cell death is several 10 μ M (see Yankner, b.a., et al., Science, 250, 279-282(1990)) which is more than 1000 times higher than the concentration of amyloid β present in the brain of alzheimer's disease patients; (2) in the brain of an alzheimer's disease patient, the deposition amount of β -amyloid fibrils is not necessarily related to the degree of disorder of higher functions such as memory or cognitive function, and even if a large amount of β -amyloid fibrils is deposited, there are cases where no clinical symptoms are exhibited; (3) furthermore, the deposition site of amyloid β in the brain does not necessarily coincide with the site of neuronal cell shedding; (4) learning behavior abnormalities also occur in the brain of mice overexpressing APP, before or without deposition of beta amyloid fibers; (5) the content of soluble beta amyloid in the brain of the Alzheimer disease patient is increased by more than 10 years before the deposition of the insoluble fiber; these facts suggest that the true cause of the toxicity of beta-amyloid is not beta-amyloid fibrils.

The present inventors have previously proposed a highly toxic self-associated amyloid β -containing solution for inducing cell death in nervous system cells at a concentration equivalent to that of self-associated amyloid β present in the body of a patient suffering from Alzheimer's disease or the like, and a method for producing the same (Japanese patent application laid-open No. 2001-247600). Further, a method for isolating the true cause of the neurocytotoxicity contained in the self-associating type β -amyloid-containing liquid was also found, and it is known from the results of the analysis that the true cause is self-associating type β -amyloid having a granular form with a particle diameter of about 10 to about 20nm and is named amyloid sphere (amylosphere) (see Hoshi, m., et al, proc. natl. acad. sci. u.s.a., 100, 6370-6375 (2003)). In the present specification, according to the name, self-associated β amyloid having a granular form with a particle size of about 10 to about 20nm is referred to as "amyloid sphere".

Since amyloid spheres induce nerve cell death at a concentration equivalent to that of beta amyloid existing in the brain of alzheimer's disease patients, and phosphorylation of tau protein, which is another pathological marker, is caused during nerve death by amyloid spheres, and is consistent with the symptoms caused by alzheimer's disease, it is considered that the amyloid spheres are a true cause of toxicity of beta amyloid in the brain. Therefore, if an antibody that (1) inhibits the formation of amyloid spheres or (2) inhibits the toxicity of amyloid spheres to nervous system cells is obtained, it can be used as a therapeutic or prophylactic agent for alzheimer's disease. In addition, if (3) an antibody having higher reactivity to amyloid spheres than to amyloid precursor protein, amyloid β monomer, amyloid β fibrils, and the like is obtained, the antibody can be applied to a measurement for diagnosing alzheimer's disease.

The method for producing an antibody using an amyloid sphere as an antigen may be a known method. In addition, rabbit polyclonal anti-amyloid sphere antibodies (ASD2, ASD3) and mouse monoclonal anti-amyloid sphere antibodies (MASD1, MASD2, MASD3) (WO 2006/016644) (antibodies that react with amyloid spheres are sometimes referred to as "anti-amyloid sphere antibodies") have been obtained. However, no antibody has been obtained which has low reactivity with amyloid precursor protein, specifically reacts with amyloid spheres, and inhibits toxicity of the protein to nervous system cells. Among them, rabbit polyclonal anti-amyloid spheroid antibodies (ASD2, ASD3) and mouse monoclonal anti-amyloid spheroid antibodies (MASD1, MASD2, MASD3) described in WO2006/016644 are respectively shown below in the specification of the present application.

ASD2→rpASD2

ASD3→rpASD3

MASD1→mASD1

MASD2→mASD2

MASD3→mASD3

Non-patent document 1: selkoe, D.J., Annu.Rev.Neurosci, 12, 463-490(1989)

Non-patent document 2: glenner, G.G.and Wong, C.W., biochem. biophysis. Res. Commun, 120(3), 885-890(1984)

Non-patent document 3: ihara, Y.et., J.biochem., 99, 1807-1810(1986)

Non-patent document 4: Grundke-Iqbal, I.et al, Proc.Natl.Acad.Sci.USA, 83, 4913-

Non-patent document 5: lorenzo, A.and Yankner, B.A., Proc.Natl.Acad.Sci.USA, 91, 12243-

Non-patent document 6: yankner, B.A., et.al., Science, 250, 279-Asonic 282(1990)

Non-patent document 7: hoshi, m., et. al., proc.natl.acad.sci.u.s.a., 100, 6370-6375(2003)

Patent document 1: japanese laid-open patent publication No. 2001-247600

Patent document 2: WO No. 2006/016644

Disclosure of Invention

The purpose of the present invention is to obtain an antibody that has higher reactivity to amyloid spheres than to amyloid precursor protein, higher reactivity to amyloid spheres than to beta-amyloid fibrils or beta-amyloid monomeric protein, or an antibody that has higher reactivity to amyloid spheres than to amyloid precursor protein and that has activity to inhibit induction of neuronal cell death by amyloid spheres. Further, another object of the present invention is to provide a method for screening a therapeutic and/or prophylactic agent for alzheimer's disease using the above antibody, and a method for detecting an individual with alzheimer's disease. It is another object of the present invention to provide a neurocyte protective agent using the above antibody, a reagent for detecting alzheimer's disease, and a pharmaceutical product such as a therapeutic and/or prophylactic agent for alzheimer's disease. It is another object of the present invention to provide a solid phase carrier for detecting the antibody. It is another object of the present invention to provide a hybridoma producing the antibody.

The present inventors have intensively studied to solve the above problems, and as a result, have found that the following antibodies have low reactivity to amyloid precursor protein, higher reactivity to amyloid spheres than to β -amyloid fibrils or β -amyloid monomer protein, and further have activity of inhibiting induction of neuronal cell death by amyloid spheres: the antibody is a monoclonal antibody produced by subcutaneously immunizing a hamster with an amyloid sphere, constructing a hybridoma from spleen cells obtained from the animal, and producing the hybridoma. In addition, it was found that the antibody has low cross-reactivity with human normal tissues and specifically reacts with Alzheimer's brain. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.

(1) An antibody which has higher reactivity to an amyloid spheroid than to an amyloid precursor protein and has any one of the following characteristics of 1 or more.

(i) The reactivity to amyloid spheres is higher than that to beta amyloid fibrils;

(ii) the reactivity to amyloid spheres is higher than that of beta amyloid monomeric protein;

(iii) has activity of inhibiting the induction of nerve cell death by amyloid spheroids.

(2) The antibody according to (1), wherein the reactivity of the antibody to amyloid spheres is 3 times or more higher than that to β amyloid fibrils in a system in which the reactivities of the antibody to amyloid spheres and β amyloid fibrils are compared using the same antibody concentration and antibody amount and the same antigen protein concentration and antigen protein amount.

(3) The antibody according to (1) or (2), wherein in a system in which the reactivity of the antibody to amyloid spheres and β amyloid fibrils is compared using the same antibody concentration and antibody amount and the same antigen protein concentration and antigen protein amount, the reactivity of the antibody to amyloid spheres is 5 times or more the reactivity to β amyloid fibrils.

(4) The antibody according to any one of (1) to (3), wherein in a system in which the reactivity of the antibody to amyloid spheres and beta-amyloid monomer protein is compared using the same antibody concentration and antibody amount and the same antigen protein concentration and antigen protein amount, the reactivity of the antibody to amyloid spheres is 50 times or more the reactivity to beta-amyloid monomer protein.

(5) The antibody according to any one of (1) to (4), wherein in a system in which the reactivity of the antibody to amyloid spheres and beta-amyloid monomer protein is compared using the same antibody concentration and antibody amount and the same antigen protein concentration and antigen protein amount, the reactivity of the antibody to amyloid spheres is 500 times or more the reactivity to beta-amyloid monomer protein.

(6) The antibody according to any one of (1) to (5), wherein the antibody is obtained using an amyloid sphere as an antigen.

(7) The antibody according to any one of (1) to (6), wherein the antibody is a monoclonal antibody.

(8) The antibody according to (7), wherein the dissociation constant of the antibody for an amyloid sphere is 10-9 or less.

(9) The antibody according to any one of (1) to (8), which specifically reacts with the Alzheimer's disease brain without showing significant cross-reactivity to human normal tissues.

(10) The antibody according to any one of (1) to (9), wherein the antibody recognizes a stereostructurally specific epitope of an amyloid sphere.

(11) The antibody according to any one of (1) to (10), wherein the antibody is a hamster-derived antibody.

(12) The monoclonal antibody according to any one of (1) to (11), wherein the antibody is produced by a hybridoma having a deposit number of FERM BP-10871 or FERM BP-10872.

(13) A humanized antibody obtained by humanizing a hamster monoclonal antibody produced from a hybridoma deposited with the FERM BP-10871 or FERM BP-10872 accession number.

(14) The humanized antibody or a fragment thereof of (13), which comprises a humanized heavy chain and a humanized light chain,

the humanized heavy chain comprises 3 heavy chain Complementarity Determining Regions (CDRs) derived from a hamster monoclonal antibody produced by the hybridoma having the deposit number FERM BP-10872, and a heavy chain variable region framework sequence derived from a human immunoglobulin heavy chain;

the humanized light chain comprises 3 light chain Complementarity Determining Regions (CDRs) derived from a hamster monoclonal antibody produced by the hybridoma having the deposit number FERM BP-10872, and a light chain variable region framework sequence derived from a human immunoglobulin light chain;

and, 3 heavy chain Complementarity Determining Regions (CDRs) have the following amino acid sequences, respectively:

heavy chain CDR 1: asp Tyr Phe Met Ser (serial number 11);

heavy chain CDR 2: gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe GlySer Val Lys Gly (SEQ ID NO: 12); and

heavy chain CDR 3: asn Arg Glu Val Gly Gly Leu Asp Asn (serial No. 13);

the 3 light chain Complementarity Determining Regions (CDRs) have the following amino acid sequences, respectively:

light chain CDR 1: thr Leu Arg Ser Gly Ile Ser Val Gly Gly Lys Asn Ile Tyr (serial number 14);

light chain CDR 2: tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro (serial number 15); and

light chain CDR 3: ser Ile His Glu Ser Asn Ala Tyr Val (serial number 16).

(15) The humanized antibody or a fragment thereof of (13), which comprises a humanized heavy chain variable region and a light chain variable region;

the humanized heavy chain variable region comprises the following amino acid sequence (SEQ ID NO: 17):

wherein Xaa at position 49 is Gly or Ala, and Xaa at position 81 is Leu or Val; and Xaa at position 100 is Thr or Arg;

the light chain variable region comprises the following amino acid sequence (SEQ ID NO: 18):

in the formula, Xaa at position 2 is Ser or Ala, Xaa at position 8 is Ser or Ala, Xaa at position 48 is Tyr or Phe, Xaa at position 49 is Leu or Phe, Xaa at position 51 is Lys, Phe or Arg, Xaa at position 74 is Ala or Thr, and Xaa at position 79 is Gly or Ala.

(16) The humanized antibody or a fragment thereof according to (15), which has a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 5 and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 7.

(17) A screening method for a therapeutic and/or prophylactic agent for alzheimer's disease, which comprises: a test substance and the antibody according to any one of (1) to (16) are contacted with an amyloid sphere, and a candidate substance is selected using the binding property of the test substance to the amyloid sphere as an index.

(18) A method of detecting an individual with alzheimer's disease comprising: contacting a biological sample obtained from an individual suspected of having Alzheimer's disease with the antibody according to any one of (1) to (16), and determining the presence or absence of a substance that reacts with the antibody in the sample.

(19) A neuroprotective agent comprising the antibody according to any one of (1) to (16).

(20) A reagent for detecting Alzheimer's disease, which comprises the antibody according to any one of (1) to (16).

(21) A pharmaceutical product comprising the antibody according to any one of (1) to (16).

(22) A therapeutic and/or prophylactic agent for Alzheimer's disease, which comprises the antibody according to any one of (1) to (16).

(23) A solid phase carrier for detecting the antibody according to any one of (1) to (16), wherein the solid phase carrier is formed by coating an amyloid sphere.

(24) A hybridoma producing the antibody of (7) or (8).

(25) Hybridomas having a deposit number of FERM BP-10871 or FERM BP-10872.

(26) A nucleic acid comprising a sequence encoding the heavy chain or light chain of the humanized antibody according to any one of (13) to (16), or a fragment thereof.

(27) An expression vector for expressing the humanized antibody or fragment of any one of (13) to (16), which comprises a nucleotide sequence encoding the antibody or fragment.

The antibody of the present invention has low reactivity to amyloid precursor protein, higher reactivity to amyloid spheres than to β -amyloid fibrils or β -amyloid monomer protein, and has an activity of inhibiting induction of neuronal cell death by amyloid spheres, and therefore, can be used as a therapeutic or prophylactic agent for alzheimer's disease, and can also be used for detection of alzheimer's disease individuals.

Drawings

FIG. 1 is a graph showing the results of an amyloid sphere solid phase ELISA obtained by analyzing the reactivity of a monoclonal anti-amyloid sphere-specific antibody of the present invention.

FIG. 2 is a graph showing the results of dot blotting obtained by analyzing the reactivity of the monoclonal anti-amyloid sphere-specific antibody of the present invention.

FIG. 3 is a graph showing the results obtained by analyzing the epitope of the monoclonal anti-amyloid sphere-specific antibody of the present invention.

Fig. 4 is a graph showing the results obtained from the activity of the monoclonal anti-amyloid sphere-specific antibody of the present invention to inhibit the induction of neuronal cell death by amyloid spheres.

FIG. 5 is a graph showing the results of Western blotting obtained by analyzing the reactivity of a commercially available antibody (6E 10).

Fig. 6 is a graph showing the results of Western blotting obtained by analyzing the reactivity of mouse monoclonal anti-amyloid sphere antibodies (mASD1, mASD2, mASD 3).

FIG. 7 is a graph showing the results of Western blotting obtained by reactivity of hamster monoclonal amyloid sphere-specific antibody of the present invention.

FIG. 8 is a graph showing the reactivity of representative mouse monoclonal antibody and hamster monoclonal antibody to a human normal tissue group.

Fig. 9 is an electron micrograph showing the specificity of the antibody to fibrillar amyloid spheres and ASPD.

FIG. 10 is a diagram showing the DNA sequence and amino acid sequence of the heavy chain variable region of humanized antibody RHA/RLA (huaSD2) and the positions of CDRs 1, 2 and 3.

FIG. 11 shows the DNA sequence and amino acid sequence of the light chain variable region of humanized antibody RHA/RLA (huaSD2) and the positions of CDRs 1, 2 and 3.

FIG. 12 shows the amino acid sequence of the heavy chain variable region of the humanized antibody RHB/RLB and the positions of CDRs 1, 2 and 3.

FIG. 13 shows the amino acid sequence of the light chain variable region of the humanized antibody RHB/RLB and the positions of CDRs 1, 2 and 3.

FIG. 14 shows the amino acid sequence of the light chain variable region of the humanized antibody RHC/RLC and the positions of CDRs 1, 2, and 3.

Fig. 15 is a diagram showing the positions of CDRs 1, 2, 3 in comparison to the amino acid sequences of the heavy and light chains of three humanized antibodies obtained by the present invention. The heavy chains of RHA/RLA (huaSD2), RHB/RLB and RHC/RLC are denoted ADS2RHA, ADS2RHB and ASD2RHC, respectively. Among them, the ASD2RHA is omitted because it is the same sequence as the ADS2 RHA. The light chains of RHA/RLA (huaSD2), RHB/RLB, and RHC/RLC are denoted as ADS2RLA, ADS2RLB, and ASD2RLC, respectively.

Fig. 16 is a graph showing the inhibitory effect of the huASD2 antibody on the induction of apoptosis by DF-ASPD.

Fig. 17 is a graph showing the effect of haASD2 and huASD2 antibodies in inhibiting the induction of DF-ASPD on cell death.

Detailed Description

The antibody of the present invention has higher reactivity to amyloid spheres than to amyloid precursor protein, and has any of the following features of 1 or more (hereinafter, these may be referred to as "anti-amyloid sphere-specific antibodies").

The present invention also relates to a method for screening a drug for treating and/or preventing Alzheimer's disease using the antibody, a method for detecting an individual with Alzheimer's disease, a pharmaceutical product such as a drug for treating and/or preventing Alzheimer's disease, and a hybridoma producing the antibody. The following detailed description is given of the embodiments of the present invention, but the following description is only an example (representative example) of the embodiments of the present invention, and the scope of the present invention is not limited to these contents.

(1) Anti-amyloid spheroid specific antibodies

The anti-amyloid spheroid specific antibody of the present invention is characterized by having higher reactivity to amyloid spheroid than to amyloid precursor protein, and is represented by the following scheme.

According to a first mode, the anti-amyloid sphere-specific antibody of the present invention has higher reactivity to amyloid spheres than to β -amyloid fibrils. "reactivity to amyloid spheres" means reacting with amyloid spheres formed by the method described below. The reactivity of the antibody can be measured by a method generally used per se, and when the antibody is measured by these methods, the antibody is included in the antibody of the present invention if the antibody has higher reactivity with amyloid spheres than with β -amyloid fibrils. According to a preferred embodiment, the antibody is more than 3 times, more preferably more than 4 times, and most preferably more than 5 times more reactive with amyloid beta fibrils. In this case, the reactivity can be compared when the same antibody concentration and amount, and the same antigen protein concentration and amount are used. In addition, an antibody characterized by specifically reacting with an amyloid sphere and not reacting with a β -amyloid fibril is also included in the anti-amyloid specific antibody of the present invention.

According to a second mode, the anti-amyloid sphere-specific antibody of the present invention has higher reactivity to amyloid spheres than to amyloid beta monomeric protein. In this case, the reactivity of the anti-amyloid sphere-specific antibody to amyloid spheres is preferably 50 times or more, more preferably 100 times or more, and most preferably 500 times or more, the reactivity to amyloid beta monomeric protein (a β). In this case, the reactivity can be compared when the same antibody concentration and amount, and the same antigen protein concentration and amount are used.

When an anti-a β antibody is administered in an alzheimer disease model animal or a clinical trial, induction of cerebral hemorrhage can be confirmed. This bleeding is thought to be caused by an inflammatory response resulting from the binding of antibodies to cerebrovascular amyloid, and is a side effect of treatment with anti-a β antibodies. Cerebrovascular Amyloid deposits are seen in 80-90% of patients with alzheimer's disease and are known as Amyloid Angiopathy of the a β type (CAA). Unlike amyloid of senile plaques in alzheimer's disease, which contains a β 42as a main component, deposition in CAA is mainly caused by a β 40. Accordingly, an antibody that selectively reacts with amyloid spheres and has low reactivity with a β 40 is particularly preferable as the antibody of the present invention. Specifically, the anti-amyloid sphere-specific antibody of the present invention preferably has 50-fold or more, more preferably 100-fold or more, and most preferably 500-fold or more reactivity with amyloid spheres, as compared with a β 40.

The present invention discloses "amyloid spheres" exhibiting high reactivity with an anti-amyloid sphere-specific antibody, which is a substance having a granular morphology in which amyloid beta monomeric protein is self-associated. The "granular form" means any form as long as it is granular, and includes all of granular form, fine granular form, crystal form, agglomerated form, and the like. The particle size is usually about 10 to about 20nm, preferably about 10 to about 15nm, more preferably about 10 to about 12nm, and particularly preferably about 12 nm. The amyloid spheres have a protein concentration of about 1. mu.g/ml or less, preferably about 0.45. mu.g/ml or less, and have a high neuronal death activity of inducing cell death in cells of the nervous system. When amyloid spheres having the above-described physical properties are fractionated by glycerol density gradient centrifugation, a fraction having a glycerol concentration of about 15% or more can be obtained.

Examples of the method for measuring the reactivity of the anti-amyloid sphere-specific antibody of the present invention with an antigen include a known immunological measurement method such as an immunoblotting method, a dot blotting method, and an ELISA method, and a method of observation using an electron microscope. In addition, the amyloid β monomer as a comparative control in this case refers to a protein composed of about 40 amino acid residues, which is produced from Amyloid Precursor Protein (APP) in the body through a protease-mediated process. Various types of proteases are known depending on the type of the protease and the modification to be carried out thereafter, but the length of the C-terminal amino acid residue immediately after secretion is mainly determined depending on the lengthTo the presence of amyloid beta 40 (A beta)_1-40SEQ ID NO. 1) and amyloid beta 42 (Abeta. beta.)_1-42SEQ ID NO. 2), and beta 43 amyloid protein (A.beta._1-43The sequence No. 3) is also present in a trace amount, and the amyloid beta monomer includes any of them. In addition, partial polypeptides and derivatives thereof are also included. In addition, amyloid β fibers refer to fibrous fibers formed by self-association of amyloid β, which have nerve cell death activity. Such amyloid β fibers include, for example, fibers obtained from the body or fibers made by the methods described in Lorenzo, a.et al, proc. natl.acad.sci.usa, 91, 12243-.

According to a third mode, the anti-amyloid sphere-specific antibody of the present invention is an antibody having an activity of inhibiting induction of neuronal cell death by amyloid spheres. The "induction of nerve cell death by amyloid spheres" refers to an activity of amyloid spheres produced by the above or later-described method to induce cell death in nerve cells, and the induced cell death may be either apoptosis or necrosis. The nerve cells are not particularly limited as long as they are nerve system cells, and nerve system cells derived from mammals (human, rat, mouse, monkey, pig, etc.) can be used. Neural cells obtained by inducing differentiation from ES cells or the like can also be used. Examples of the primary cultured cells include nerve cells obtained from the hippocampus, the basal forebrain, the cerebral cortex, and the like of the above-mentioned animals. Also included are cells obtained by culturing organs such as hippocampus of the above-mentioned animals. The anti-amyloid spheroid specific antibody having such an activity is characterized by, for example, higher reactivity with amyloid spheroids than with amyloid precursor protein, amyloid β fibrils, or amyloid β monomeric protein. Among them, an anti-amyloid sphere-specific antibody having a reactivity to amyloid spheres about 10 to about 20 times as high as that to amyloid precursor protein can be preferably used.

The activity of the anti-amyloid spheroid specific antibody of the present invention to inhibit induction of neuronal cell death means preferably the ability to completely inhibit the aforementioned induction of neuronal cell death by amyloid spheroid, but it also includes the case where the antibody is partially inhibited depending on the amount of antibody to be administered. The specific method for measuring the inhibitory activity is as described later.

In addition, in addition to any 1 or more of the first to third embodiments, an antibody that specifically reacts with the alzheimer's disease brain without showing significant cross-reactivity to human normal tissues is also included in the anti-amyloid sphere-specific antibody of the present invention.

In any 1 or more embodiments of the first to third aspects or the aforementioned embodiments, an antibody that recognizes a steric-structure-specific epitope of an amyloid sphere is also included in the anti-amyloid sphere-specific antibody of the present invention. Among them, preferred is an anti-amyloid spheroid specific antibody which recognizes the N-terminus of the amyloid beta monomer protein as an epitope or does not recognize the N-terminus of the amyloid beta monomer protein as an epitope in the order listed on the amyloid beta monomer protein. The "antibody characterized by recognizing a stereostructurally specific epitope of an amyloid sphere" is specifically an antibody that can bind to an amyloid sphere in a natural state and cannot bind to an amyloid sphere in a modified state.

The specific production method of the anti-amyloid sphere-specific antibody of the present invention and the method for analyzing the above-described characteristics will be described in detail below.

(2) Preparation of amyloid protein spheres (antigens)

The antibody of the present invention can be obtained using an amyloid sphere having the following properties as an antigen. In the present invention, an amyloid sphere refers to a substance in which β amyloid is self-associated and has a granular form. The "granular form" means any form as long as it is granular, and includes all of granular form, fine granular form, crystal form, agglomerated form, and the like. The particle size is usually about 10 to about 20nm, preferably about 10 to about 15nm, more preferably about 10 to about 12nm, and particularly preferably about 12 nm. The amyloid spheres have a high nerve cell death activity to induce cell death of nervous system cells at a protein concentration of about 1. mu.g/ml or less, preferably about 0.45. mu.g/ml or less. When amyloid spheres having the above-described physical properties are fractionated by glycerol density gradient centrifugation, a fraction having a glycerol concentration of about 15% or more can be obtained.

Such amyloid spheres can be prepared by first convecting an aqueous solution containing amyloid β (first step). In addition, in order to prepare a solution containing amyloid spheres efficiently, a method of classifying amyloid spheres in an aqueous solution obtained by convection (second step) may be used. The antigen of the antibody of the present invention may be any of the above-described amyloid sphere-containing solutions.

By "amyloid beta" is meant a protein consisting of about 40 amino acid residues, which is produced in the body by a protease-mediated process from Amyloid Precursor Protein (APP). It is known that various kinds of proteases exist depending on the kind of the protease and the post-modification thereof, but β 40 amyloid (a β) mainly exists depending on the length of the C-terminal amino acid residue immediately after secretion_1-40SEQ ID NO. 1) and amyloid beta 42 (Abeta. beta.)_1-42SEQ ID NO: 2) and a trace amount of beta 43 amyloid (A.beta._1-43And sequence number 3). For the preparation of amyloid spheres, it is preferable to use, for example, a β, which is a full-length molecular species of β amyloid immediately after secretion_X-40、Aβ_X-42Or Abeta_X-43Or a mutant or derivative thereof, wherein A.beta.is particularly preferred_1-40Or Abeta_1-42. The amyloid β can be any protein such as a protein synthesized by a peptide synthesizer or the like, a commercially available protein, or a protein extracted and purified from a biological sample. When synthetic peptides are used as amyloid beta, the methods for synthesizing and extracting and purifying the synthetic peptides may be those known per se and commonly used methodsThe method is carried out. The purification degree of the synthetic peptide is sufficient to be such an extent that a single peak can be obtained by high performance liquid chromatography, and for example, gel filtration, high performance liquid chromatography, or the like can be used as a purification method. In the present specification, "amyloid β" may be referred to as "amyloid β monomer", "amyloid β 0 monomeric protein", "a β" or "a β monomer". The amyloid β thus obtained is dissolved in, for example, sterilized purified water, and used for preparation of an amyloid sphere-containing solution. The amount of the sterilized purified water to be used for dissolution may be in the range of dissolving beta-amyloid, but the concentration of beta-amyloid in the aqueous solution is preferably in the range of about 50nM to about 2mM, preferably about 1. mu.M to about 1mM, and more preferably about 50 to about 700. mu.M. The solution is preferably adjusted to an appropriate salt concentration. The salt concentration may be any concentration as long as the amyloid β can be dissolved, and for example, the final pH is about 3 to about 11, preferably about 5.5 to about 8.5, and more preferably about 7.5, and the salt is preferably about 1M or less. As a method for adjusting such a salt concentration, for example, a method of adding PBS (-) and an equal amount of an aqueous solution of β -amyloid can be used. The method of dissolution is not particularly limited as long as the amyloid β is completely dissolved in a solution having an appropriate salt concentration.

The first step of the method for producing the amyloid sphere-containing solution includes, for example, the step described in Japanese patent application laid-open No. 2001-247600. The amyloid sphere-containing solution thus obtained has an activity of inducing neuronal cell death without any treatment and can be used as the antigen of the present invention, but fractionation as the second step can yield a fraction having a higher neuronal cell death activity. As a method of classification, for example, the method described in japanese patent application laid-open No. 2002-105099 can be used. The thus obtained amyloid sphere-containing solution can be used as an antigen in the following immunization step after being subjected to treatment such as concentration, if necessary.

Examples of the method for confirming the formation of amyloid spheres include the following method for analyzing the neuronal death activity, and a method for measuring the activity by an electron microscope. The measurement method by an electron microscope may be any method as long as it can analyze the particle size of the amyloid spheres and observe the self-association of the amyloid spheres without damaging them. Specifically, for example, distilled water of 30 to 40 ℃ is first added to a glass dish having a diameter of about 18mm, about 30. mu.l of a solution of collodion 1.5% (W/V) isoamyl acetate or the like is dropped on the water surface, and the solvent is immediately evaporated to form a film. After the support film was applied to a grid and dried, carbon was evaporated in vacuum and the surface was hydrophilized using a hydrophilization treatment apparatus using glow discharge. Next, the grid on which the support membrane was attached was placed face down, and brought into contact with a droplet of the solution containing the prepared amyloid spheres, and immediately excess moisture was wiped off with filter paper, and a uranium acetate solution was added for observation. The following methods are preferred, and the like: the specimen is accelerated at a high voltage of 100 to 120kV under a stable condition using an electron microscope, and in order to prevent the specimen from being damaged by an electron beam, astigmatism correction is performed using an end portion of a grid or the like, and then observation is performed using an electron beam damage reduction method.

(3) Preparation of antibody with amyloid protein sphere as antigen

The method for obtaining an antibody using an amyloid sphere as an antigen described in (2) above is not particularly limited as long as it can obtain an antibody having higher reactivity with an amyloid sphere than with an amyloid precursor protein and having any one of the following features of 1 or more. Specifically, the method described in detail below can be preferably used.

The antigen is obtained by binding or polymerizing the amyloid spheres described in (2) above to a protein or polymer such as KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), OVA (ovalbumin) which is a common carrier, and is used as an antigen for immunization. Alternatively, a plurality of antigens prepared by a binding method with different carriers may be mixed to prepare an antigen for immunization.

The animal used for immunization is not particularly limited, and rabbit, goat, sheep, hamster, mouse, rat, guinea pig, chicken, animals other than human producing human antibody, and the like can be used. Hamsters are preferably used. After complete mixing of Freund's complete adjuvant or Freund's incomplete adjuvant with an antigen for immunization, an animal to which the antigen for immunization is administered is inoculated subcutaneously, intramuscularly or intraperitoneally. The vaccination is performed 1 time every 2 to 5 weeks until the reactivity of the antibody of the immunized animal to the vaccinated antigen is sufficiently increased. The amount of the antigen to be immunized 1 time is not particularly limited as long as the reactivity of the antibody of the immunized animal can be sufficiently increased, and specifically, is preferably about 1 to about 100. mu.g. In addition, it is preferable to repeat the collection of blood from an immunized animal and the detection of the reactivity of an antibody contained in the blood to an antigen by the method described later until the reactivity to an amyloid sphere is higher than that to a β amyloid monomer protein. Specifically, the number of times is preferably 5 to 20.

After 7 to 10 days from the last immunization, blood, ascites, and the like were collected from the animal. Preferably, whole blood is collected and serum is prepared by centrifugation or the like. The method for measuring the reactivity of the anti-amyloid sphere-specific antibody of the present invention contained in the serum may be any method as long as it can analyze the reactivity with the amyloid sphere prepared in the above (2), and examples thereof include a method in which the amyloid sphere is labeled with a fluorescent substance or the like, reacted with the serum, and then the activity of a labeling agent bound to the antibody is measured. Specifically, the method of observation under an electron microscope, an enzyme immunoassay such as ELISA described later, an immunoblotting method, a dot blotting method, or the like can be mentioned. Among these, the anti-amyloid sphere-specific antibody of the present invention can be preferably used in a method of observation under an electron microscope when measuring and comparing the reactivity with the β -amyloid fiber, and in a method of measuring and comparing the reactivity of the β -amyloid monomeric protein with the amyloid sphere as its self-associated complex, an enzyme immunoassay such as dot blot or ELISA method can be preferably used. In addition, by comparing the beta amyloid fiber, beta amyloid monomer protein or their partial polypeptide and the specific reaction of the antibody reactivity, can selectively obtain the invention of anti amyloid spheroid specific antibody.

The antibody can be isolated and purified by a known isolation and purification method for immunoglobulin. Specifically, examples thereof include a salting out method, an alcohol precipitation method, an isoelectric point precipitation method, an electrophoresis method, an adsorption method using an ion exchanger, a super centrifugation method, a gel filtration method, and a method of adsorbing and separating only a specific antibody using an antigen-antibody conjugate or an active adsorbent.

The antibody thus produced is a polyclonal antibody, which may contain IgG as a main component and other immunoglobulins such as IgM and IgA.

On the other hand, when a monoclonal antibody is prepared, only amyloid spheres as a common antigen are intravenously injected into the immunized animal, and 2 to 5 days, preferably 3 days later, spleen or lymph nodes thought to contain antibody-producing cells are collected, and the spleen cells or lymph cells are subjected to cell fusion with tumor cells. Thereafter, the antibody-producing cells (hybridomas) which have been subjected to cell fusion and immortalization are separated. The tumor cells used herein are preferably of the same species as spleen cells or lymphocytes generally prepared from immunized animals, but cells between xenogeneic animals may be used. Further, as the method of immortalization, a known method other than cell fusion may be used. For example, transformation using EB virus (Epstein-Barr virus) (D.Kozbor et al, Eur J Immunol, 14: 23(1984)) can also be used.

As examples of tumor cells, myeloma cells such as P3(P3/x63-Ag8), P3U1, NS-1, MPC-11, SP2/0-Ag14, FO, x63.6.5.3, S194, and R210 are used. The cell fusion can be carried out by a conventional method, for example, the method described in mono ク experiments with ン antibody experiment マニユアル (published by , サイエンテイフイツク 1987), G.KOHLERANDC.MILSTEIN, Nature, 256, 495(1975), etc. Cell fusion can be carried out by adding a cell fusion promoter to the fusion medium in which the cells to be fused are suspended. Examples of the cell fusion promoter include Sendai virus and polyethylene glycol having an average molecular weight of 1000 to 6000. In this case, in order to further improve the fusion efficiency, an auxiliary agent such as dimethyl sulfoxide or a cytokine such as IL-6 may be added to the fusion medium. For example, about 1 to about 10 times the amount of the spleen cells or lymphocytes to the tumor cells can be used as the mixing ratio of the tumor cells to the immunized spleen cells or lymphocytes.

As the fusion medium, various general media such as ERDF medium, RPMI-1640 medium, MEM medium, GIT medium and the like can be used, and serum such as Fetal Bovine Serum (FBS) can be removed from the medium in advance in the fusion. The fusion was performed by the following method: a predetermined amount of the immunized spleen cells or lymphocytes and tumor cells are thoroughly mixed in the culture medium, and a polyethylene glycol solution heated to about 37 ℃ in advance is added to about 20 to about 50%, and the mixture is preferably reacted at about 30 to 37 ℃ for about 1 to 10 minutes. Thereafter, the operation of adding an appropriate medium one by one and centrifuging to remove the supernatant was repeated.

The target hybridoma is cultured in a usual selective medium such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). In order to cause the death of cells (unfused cells and the like) other than the target hybridoma, the culture in the HAT medium may be performed for a sufficient period of time, usually several days to several weeks.

The antibody produced by the hybridoma obtained is contained in the culture supernatant of the hybridoma. Hybridomas that produce the anti-amyloid sphere-specific antibody of the present invention can be selectively obtained by measuring the reactivity, reaction specificity, and the like of the antibody in the same manner as the method for measuring the polyclonal antibody.

The obtained hybridoma is cloned by the limiting dilution method, whereby a hybridoma clone that produces a single monoclonal antibody can be obtained. This hybridoma clone is cultured in a medium containing about 1 to about 10% of FBS from which bovine antibodies (IgG) contained in FBS have been removed in advance or a serum-free medium, and the obtained culture supernatant is used as a raw material for purifying the target monoclonal antibody. On the other hand, the obtained hybridoma clone may be transplanted into the abdominal cavity of a Balb/c mouse or a Balb/c (nu/nu) mouse to which pristane has been previously administered, and ascites containing a monoclonal antibody at a high concentration may be collected 10 to 14 days later and used as a raw material for purifying the target monoclonal antibody. The method for purifying a monoclonal antibody can be carried out by using a common immunoglobulin purification method such as ammonium sulfate fractionation, polyethylene fractionation, ethanol fractionation, anion exchange chromatography, affinity chromatography in which protein a, protein G, an anti-mouse immunoglobulin antibody or the like is bound, and the like.

The thus obtained anti-amyloid sphere-specific antibody of the present invention may be used as it is, or may be used in the form of Fab obtained by papain treatment or in the form of F (ab') obtained by pepsin treatment, which is a predetermined method₂Or Fab' form. In addition, fragments containing Complementarity Determining Regions (CDRs) in both the H chain and L chain of the antibody, hypervariable regions, and the like, or genes encoding the same, are obtained by methods known per se, and furthermore, human-type antibodies are also included in the anti-amyloid sphere-specific antibody of the present invention. In addition, a fully human antibody prepared by phage display method or human antibody-producing mouse or the like is also included in the anti-amyloid sphere-specific antibody of the present invention.Furthermore, the above hybridoma cell lines producing monoclonal antibodies are also encompassed by the present invention. Specific examples of the hybridoma of the present invention include those obtained in the following examples and having a deposit number of FERM BP-10871 or FERM BP-10872.

An antibody (hereinafter referred to as a "humanized antibody") obtained by humanizing a non-human (mouse, rat, hamster, rabbit, etc.) antibody is a chimeric immunoglobulin, an immunoglobulin chain or a fragment thereof (e.g., Fv, Fab ', F (ab')₂Or an antigen-binding subsequence other than an antibody) comprising a minimal sequence derived from a non-human immunoglobulin. Particularly preferred as the humanized antibody is an antibody composed partially or entirely of an amino acid sequence derived from a germ line of a human antibody by modifying the sequence of an antibody having a Complementarity Determining Region (CDR) of a non-human antibody. This change is achieved by replacing the non-human antibody constant region with the human antibody constant region, enabling the production of human/non-human chimeras with a tolerable degree of low immunogenicity for pharmaceutical applications. More preferably, even the variable regions and CDRs of the antibody are humanized by techniques known in the art to date. The framework regions of the variable regions are sometimes replaced with corresponding human framework regions, with the non-human CDRs being substantially unchanged or replaced by sequences derived from their human genome.

A humanized antibody is further an antibody comprising a human framework and at least 1 non-human antibody-derived CDR, where the presence of any constant region is meant to be substantially identical to a human immunoglobulin constant region. Substantially the same means that at least 85 to 100%, preferably 95 to 100%, of the amino acid sequences are the same. That is, all parts of the humanized antibody except the CDR portions are the same as those corresponding to 1 or more natural human immunoglobulin sequences.

Humanized antibodies have at least 3 advantages over non-human and chimeric antibodies when used as pharmaceuticals for human therapy.

1) Since the effector is partially human, it interacts well with other factors in the immune response in the human body. For example, target cells are efficiently destroyed by Complement Dependent Cytotoxicity (CDC) or Antibody Dependent Cellular Cytotoxicity (ADCC).

2) The human immune system is thought not to recognize the framework or constant regions of the humanized antibody as a foreign object. Therefore, it is considered that when the humanized antibody is administered into a human body, the antigen-antibody reaction is lower than that of a non-human antibody or a chimeric antibody.

3) It has also been reported that non-human antibodies are administered with a shorter half-life than human antibodies in the human circulatory system. When a humanized antibody is administered, it is expected to have substantially the same half-life as a natural human antibody, and the dose and frequency of administration can be reduced.

Methods for humanizing non-human antibodies are well known in the art. For example, the method can be carried out by the method of Winter et al (Japanese patent No. 2912618), the method of Jones et al (Nature, 321: 522(1986)), the method of Riechmann et al (Nature, 332: 323(1988)), the method of Verhoeyen et al (Science, 239: 1534(1988)), the method of Queen et al (Proc. Natl. Acad. Sci. USA 88: 2869(1991)), and the like. In the operation of obtaining a humanized antibody, silent mutation of the codon is preferably performed in order to optimize expression in a host cell expressing the humanized antibody (for example, a method of Nakamura et al: nucleic Acid Res 29: 292 (2000)). The antibody thus obtained is also encompassed by the present invention as long as it has the specificity described in the present application, and a humanized antibody characterized by having an amino acid sequence in which 1 or more amino acids are deleted, substituted, inserted or added in the variable region.

(4) Determination of the reactivity of an anti-amyloid sphere-specific antibody to an antigen

Specific examples of ELISA and dot blot methods for measuring the reactivity of the anti-amyloid sphere-specific antibody of the present invention to an antigen are described below. Examples of ELISA include solid phase ELISA and liquid phase ELISA. In addition, the dissociation constant of the anti-amyloid sphere-specific antibody of the present invention for an antigen can also be measured. The dissociation constant of the antibody can be measured by using an instrument such as BIACore (manufactured by BIACORE) or by a method according to the instrument.

(a) Solid phase carrier coated with amyloid protein spheres and amyloid protein sphere solid phase ELISA

The anti-amyloid sphere-specific antibody can be measured by measuring the reactivity of the anti-amyloid sphere-specific antibody to an antigen using a solid phase carrier coated with amyloid spheres. Examples of the solid phase carrier include spherical, rod-like, plate-like carriers made of plastic such as polystyrene and polypropylene, and preferably plastic plates. For coating the solid phase carrier with the amyloid spheres, a common method such as an adsorption method or a method using a crosslinking agent can be used, and an adsorption method in which the amyloid spheres are physically adsorbed is preferably used from the viewpoint of simplicity.

Specifically, an amyloid sphere ELISA is an assay method using a solid phase carrier coated with an amyloid sphere. First, the amyloid spheres prepared in (2) above were coated on an ELISA plate manufactured by Nunc corporation or the like. In this case, the solvent may be any solvent as long as it does not disassociate amyloid protein spheres, but for example, PBS (-) is preferably used. The plate is washed with an appropriate solution, for example, a physiological saline solution containing a surfactant such as 0.05% Tween20, blocked with a bovine serum albumin/Phosphate Buffer Solution (PBS), and reacted with the antibody obtained above. Thereafter, after further washing, it is contacted as a second antibody (secondary antibody) with an antibody reactive with immunoglobulin of the immunized animal. After the washing, the second antibody bound to the plate is detected using the activity of the labeled substance or the like as an index. The activity of such a labeled substance can be measured, for example, using an ELISA plate reader (ELISA plate reader). In addition, using the amyloid sphere ELISA, the epitope (epitope) of the anti-amyloid sphere specific antibody of the present invention can be determined. Specifically, by amyloid sphere ELISA, the binding competition inhibition of fragments of beta amyloid monomeric protein with anti-amyloid sphere specific antibodies can be determined and the epitope determined. Fragments of amyloid beta monomeric protein may also be combined in multiple ways. Furthermore, by amyloid sphere ELISA, the binding competition inhibition of antibodies with known epitopes to anti-amyloid sphere specific antibodies can be determined and the epitope determined. Furthermore, epitopes can be identified by "Antibodies: a Laboratory Manual "(Ed Harlow et al, Cold spring harbor LABORATORY (1988)), and the like, or by following the method described therein.

(b) Amyloid spheroid liquid phase ELISA

A sample containing an antibody against amyloid spheres, such as hybridoma culture supernatant, is mixed with the amyloid spheres for more than 1 hour at room temperature and reacted. To an ELISA plate coated with an appropriate amount of rabbit anti-amyloid globulin sphere IgG in advance and blocked with, for example, bovine serum albumin/PBS, a certain amount of the above-mentioned mixture is added, and the mixture is allowed to react at room temperature for 1 hour or more. Then, after further washing, it is contacted as a second antibody with an antibody reactive with immunoglobulin in the sample, such as an anti-mouse IgG antibody, an anti-mouse IgM, an anti-mouse immunoglobulin. After the washing, the second antibody bound to the detection plate is detected using the activity of the labeled substance or the like as an index. The activity of such a labeled substance can be measured using, for example, an ELISA plate reader or the like.

(c) Beta amyloid protein monomer ELISA

A sample containing an antibody, for example, a hybridoma culture supernatant is mixed with a beta-amyloid monomer protein having biotin bound to the N-terminus or a beta-amyloid monomer protein having biotin bound to the C-terminus, and reacted at room temperature for 1 hour or more. The mixture was added to a streptavidin ELISA plate blocked with bovine serum albumin/PBS in advance, and allowed to react at room temperature for 30 minutes or longer. Then, it is contacted as a second antibody after washing with an antibody reactive with immunoglobulin in the sample, for example, an anti-mouse IgG antibody, an anti-mouse IgM, an anti-mouse immunoglobulin. After the washing, the second antibody bound to the detection plate is detected using the activity of the labeled substance or the like as an index. The activity of such a labeled substance can be measured, for example, using an ELISA plate reader or the like.

(d) Dot blotting method

An example of a specific method of dot blotting for measuring the reactivity of the anti-amyloid sphere-specific antibody of the present invention to an antigen is described below. First, an appropriate amount of the amyloid spheres prepared in (2) above was blotted on a nitrocellulose membrane or the like using a commercially available Blotter or the like manufactured by Bio Rad, and the like. In this case, the solvent may be any solvent as long as it does not disassociate the amyloid spheres, and PBS (-) is preferably used, for example. In addition to blotting of amyloid spheres, amyloid beta monomeric protein or a partial peptide thereof, and a solvent itself were also blotted as controls. The membrane is washed with an appropriate buffer such as Phosphate Buffered Saline (PBS) and blocked with skim milk/TTBS (Tween-Tris buffered saline), the membrane is contacted with the antibody obtained above, and then further washed with TTBS or the like, and then the antibody is contacted with an antibody that reacts with immunoglobulin of an immunized animal as a second antibody, and after washing the second antibody in the same manner, the second antibody bound to the membrane is detected using the activity of a labeled substance or the like as an index. As a control, an antibody reactive with amyloid-beta monomeric protein is preferably used. Examples of such an antibody include "6E 10" (manufactured by Senetek corporation).

(5) Assay for Activity of inhibiting Induction of nerve cell death by amyloid spheroids

An example of a method for analyzing the activity of the anti-amyloid spheroid specific antibody of the present invention to inhibit induction of neuronal cell death by amyloid spheroids (hereinafter, this may be referred to as "neuronal toxicity neutralizing activity" or "activity to inhibit induction of neuronal cell death") will be described below.

First, induction of nerve cell death using amyloid spheres can be performed as follows: the amyloid spheres are added to a culture solution of cells of the nervous system or the like, and cultured according to a usual method. Here, when analyzing whether the anti-amyloid sphere-specific antibody of the present invention has such a neurocytotoxicity neutralizing activity, it can be performed as follows: the above-mentioned nerve cells were cultured together with amyloid spheres in the presence of an anti-amyloid sphere-specific antibody, and it was confirmed that no induction of nerve cell death was observed. The cell death induced by amyloid spheres may be either apoptosis or necrosis. The cells to be used are not particularly limited as long as they are nervous system cells, and are preferably nervous system cells derived from mammals (e.g., human, rat, mouse, monkey, and pig). In addition, primary cultured cells are preferred. As the primary culture cells, those obtained from the hippocampus and forebrain basement, cerebral cortex, etc. of the above-mentioned animals are preferable. In addition, cells obtained by culturing organs such as hippocampus of the above-mentioned animals may be used as they are. In addition, neural cells obtained by inducing differentiation from ES cells or the like can also be used.

These cells or organs can be cultured by a usual culture method. Specifically, as a method for primary culture of cells of the nervous system and culture of established cell lines of the nervous system, methods described in Hoshi, M.et al, Proc.Natl.Acad.Sci.USA., 93, 2719-2723(1996) and Schubert, D.et al, Nature, 249(454), 224-227(1974) and the like can be used, and for organ culture, methods described in Gary Bank and Kimber Goslin, Current neural cells, 2 d edition, MIT Press, Cambridge (1998) and the like can be used. The amount of the amyloid spheres to be added is appropriately selected in order to induce cell death in the cells and organs of the nervous system thus cultured, and the amyloid spheres are usually capable of inducing cell death at a concentration substantially equal to that of toxic β amyloid present in the brain of a patient suffering from alzheimer's disease or the like. For example, the amyloid spheres obtained in (2) above can induce cell death in primary culture cells in such an amount that the amyloid β concentration in the culture solution is about 1 μ g/ml or less, more preferably about 0.45 μ g/ml or less, as described above. However, the above concentrations are merely examples and are not limited to the amounts.

The amount of the anti-amyloid sphere-specific antibody of the present invention present in the culture may be appropriately selected depending on the reactivity of the antibody used to the antigen, and specifically, is preferably present in an amount of, for example, about 0.0001 to about 1 mg/ml. The time at which the anti-amyloid sphere-specific antibody is added to the culture medium is not particularly limited as long as the neurotoxic neutralizing activity can be confirmed, and the induction of neuronal cell death by amyloid spheres is initiated from about 6 hours after the culture, and therefore, it is preferable to add the anti-amyloid sphere-specific antibody to the culture medium in the early stage of the culture. In addition, after incubating the amyloid spheres with the anti-amyloid sphere specific antibody in a separate container, it is added to the culture solution. In addition, as a control, it is preferable to use an antibody that is not reactive with amyloid spheres or an antibody that has low reactivity with amyloid spheres and whose reactivity does not affect induction of nerve cell death. As such an antibody, for example, an antibody against β amyloid monomer protein, specifically, for example, "6E 10" (manufactured by Senetek) or the like can be preferably used.

The cell death induced by amyloid spheres usually occurs from about 6 hours after the addition of an effective amount of amyloid spheres, and significant cell death can be observed after about 48 hours. Therefore, in this assay method, when the induction of nerve cell death is measured, it is preferably about 20 hours after the start of culture, but it can be appropriately selected depending on the cell death activity system of the amyloid sphere used.

As a method for measuring these nerve cell death activities, a commonly used cell death assay method can be used. Specifically, MTT activity assay (Mossman, t., j.immunol. methods, 65, 55(1983)), staining method using propidium iodide (ankacronna, m.et al, Neuron, 15, 961(1995)) or the like, or Trypan blue exclusion (Trypan blue dye exclusion) (wo, k.b., Funkhouser, w.k., Sullivan, c.and Alabaster, o., Cell tissue kinetic, 13(6), 591-. Among them, a staining method using propidium iodide or the like or an ELISA for detecting a fragmented DNA is particularly preferable. The staining method using propidium iodide or the like may be a method of singly staining propidium iodide, which selectively stains dead cells, or a method of staining propidium iodide in combination with other plural staining dyes. Specific examples of the combinable staining dye include Calcein-AM (manufactured by Molecular Probes) which selectively stains living cells, and Hoechst33258(H33258, Bisbenzimide H33258) which stains all cells.

In addition, the activity of the anti-amyloid sphere-specific antibody of the present invention to inhibit induction of neuronal cell death can also be performed by directly administering the anti-amyloid sphere-specific antibody of the present invention to an animal subject. The cell death induced by amyloid spheres may be either apoptosis or necrosis. The animal to be used is not particularly limited as long as it has nervous system cells such as mammals (mice, rats, primates, etc.), but animals, particularly those undergoing nerve cell death such as model animals of alzheimer's disease, are preferably used. In addition, the administration method may be a method of directly administering the drug to a site where nervous system cells such as brain exist, or a method used for ordinary drug administration such as oral administration, intravenous injection, and intraperitoneal administration. As a method of directly administering a drug to a site where a neural cell such as a brain exists, specifically, for example, in the case of a brain tissue such as a rat or a mouse, a method of administering a drug into a ventricle near a target site by an osmotic pump, a method of micro-perfusion (micro-perfusion) into a brain parenchyma of the target site by a micropipette or the like, and after a certain period of administration, changes in brain function are measured by PET or MRI, and then a tissue around the administration site is rapidly taken out and prepared into a tissue section, whereby the presence or absence of death of a neural cell can be examined. The presence or absence of neuronal cell death can be examined by tissue staining or Western blotting, and examples of the tissue staining include TUNEL staining and immunostaining using an anti-Caspase antibody.

(6) Therapeutic/prophylactic agent for alzheimer's disease and screening method

When amyloid spheroids are added to cultured cells of the nervous system, they can cause the cell to die, and thus it is considered that amyloid spheroids formed by self-association of β amyloid also induce neurodegeneration in alzheimer's disease.

Since the anti-amyloid sphere-specific antibody of the present invention exhibits high reactivity to the amyloid sphere and has an activity of inhibiting induction of neuronal cell death by the amyloid sphere, it is possible to screen a therapeutic and/or prophylactic agent for alzheimer's disease by binding a test substance to the amyloid sphere in competition with the anti-amyloid sphere-specific antibody of the present invention and selecting the substance using the reactivity as an index. The anti-amyloid spheroid specific antibody of the present invention can be used as an active ingredient of a therapeutic and/or prophylactic agent for alzheimer's disease. That is, the anti-amyloid sphere-specific antibody of the present invention has low reactivity with an amyloid β monomer represented by amyloid precursor protein or another structure capable of forming the monomer, and has high specificity for the brain, and therefore can be a therapeutic agent for alzheimer's disease with higher safety than the conventional anti-amyloid sphere antibody known in WO 2006/016644.

Specific examples of the method for screening the substance will be described below. Examples of the analyte include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts, and these compounds may be novel compounds or known compounds. The reactivity with amyloid spheres includes a method of analyzing the reactivity of the anti-amyloid sphere-specific antibody of (4) above with amyloid spheres, a method of adding the analyte to a reaction solution, and the like. The mixing amounts of the amyloid spheres, the anti-amyloid sphere-specific antibody, and the test substance may be selected by selecting appropriate concentrations, respectively.

The analyte is preferably labeled with a labeling substance or the like in advance. By the analysis, it can be judged that a substance that binds to amyloid spheres can be used as an active ingredient of a therapeutic/prophylactic agent for alzheimer's disease. It is also preferable to confirm whether or not the induction of neuronal cell death by amyloid spheres is inhibited by substituting a selected substance for the anti-amyloid sphere-specific antibody in the method described in (5) above.

The thus selected substance and the anti-amyloid sphere-specific antibody of the present invention are useful as an active ingredient of a drug for the prophylaxis and/or treatment of alzheimer's disease, and may be a physiologically tolerable salt, hydrate, solvate, or the like thereof. Preferably, the metal ion is added with Fe, Zn, etc., or the sugar chain or glycoprotein. Examples of physiologically acceptable salts include salts of inorganic acids such as hydrochloride and sulfate, salts of organic acids such as citrate, oxalate and p-toluenesulfonate, and salts of amino acids such as glycine. Further, an antibody specific to an amyloid sphere or a fully human antibody modified to a human type by the above method is more preferably used. The antibody can be modified to be suitable for administration to humans or the like by a combination of methods known per se.

The drug provided by the present invention contains, as an active ingredient, a substance determined to have an inhibitory effect on neuronal cell death by the screening method of the present invention or the anti-amyloid sphere-specific antibody of the present invention, and can be used as a drug for the prevention and/or treatment of alzheimer's disease. The substance determined to have an inhibitory effect on nerve cell death and the anti-amyloid sphere-specific antibody per se by the screening method of the present invention can be administered to a patient as a pharmaceutical product, but it is generally preferable to prepare a pharmaceutical composition containing 1 or 2 or more of these active ingredients and administer the pharmaceutical composition to a patient. Examples of such pharmaceutical compositions include preparations for oral administration such as tablets, capsules, granules, fine granules, powders, pills, troches (troche), sublingual preparations, and liquid preparations, and preparations for parenteral administration such as injections, suppositories, ointments, and patches.

Tablets or capsules for oral administration are usually provided as unit doses, and can be produced by adding usual carriers for pharmaceutical preparations such as binders, fillers, diluents, tabletting agents, lubricants, disintegrants, colorants, flavoring agents, and wetting agents. Tablets can be produced by coating according to a method known in the art, for example, with an enteric coating agent or the like, and using, for example, a filler, a disintegrant, a lubricant, a wetting agent or the like.

Liquid preparations for oral administration may be provided in the form of a dry preparation which can be redissolved with water or an appropriate medium before use, in addition to aqueous or oily suspensions, solutions, emulsions, syrups, elixirs and the like. The liquid preparation may contain conventional additives such as an anti-settling agent, an emulsifier, a preservative, and, if necessary, a conventional flavoring agent or coloring agent.

The preparation for oral administration can be produced by a method known in the art, such as mixing, filling, or tableting. In addition, the active ingredient may be distributed in a preparation prepared by repeating the compounding operation and using a large amount of a filler or the like. Preparations for parenteral administration are usually provided in the form of liquid carrier dosage formulations containing a substance as an active ingredient and a sterile medium. Solvents for parenteral administration are generally produced by dissolving a substance as an active ingredient in a medium, sterilizing and filtering the solution, filling the solution into a suitable vial or ampoule, and sealing the vial or ampoule. To improve stability, the composition may be frozen and filled into vials and the water removed under vacuum. The parenteral suspension can be produced by substantially the same method as that for the parenteral solution, but is preferably produced by suspending the active ingredient in a medium and sterilizing with ethylene oxide or the like. In addition, a surfactant, a wetting agent, or the like may be added as necessary in order to uniformly distribute the active ingredient.

The dose of the substance as the active ingredient can be determined appropriately in consideration of the level of activity of the substance, the purpose of therapy or prevention, the symptoms of the patient, the body weight, the age, the sex, and the like. In addition, administration is preferably performed 1 to several times per day. For example, when the anti-amyloid sphere-specific antibody of the present invention is an active ingredient, the amount thereof to be administered may be generally about 1 μ g to about 100mg, preferably about 10 μ g to about 50mg per kg body weight in 1 administration.

(7) Method and reagent for detecting Alzheimer's disease individual using anti-amyloid spheroid specific antibody

When amyloid spheres are added to cultured cells of the nervous system, the cells die, and thus it is considered that in alzheimer's disease, amyloid spheres formed by self-association of β amyloid monomeric proteins likewise induce neurodegeneration. Since the anti-amyloid sphere-specific antibody of the present invention has high reactivity to amyloid spheres, detection of amyloid spheres in a biological sample using the antibody enables detection of individuals with alzheimer's disease.

Examples of the biological sample include body fluids such as blood, cerebrospinal fluid, and urine obtained from an individual suspected of having alzheimer's disease, and blood is particularly preferable. This sample can be obtained as follows: for example, in the case of blood, blood can be collected from an elbow vein or the like of an individual suspected of having alzheimer's disease by a blood collection tube or the like, and plasma or serum can be separated by a method such as centrifugation. In the case of using cerebrospinal fluid as a sample, it can be obtained by collecting cerebrospinal fluid from an individual suspected of having alzheimer's disease by, for example, lumbar puncture under anesthesia and centrifuging the collected cerebrospinal fluid. In order to prevent amyloid spheroid changes, blood coagulation, and the like in the obtained biological sample, it is preferable to add an enzyme inhibitor to the sample at the time of sample collection or after sample collection. As the enzyme inhibitor, protease inhibitors such as aprotinin (aprotinin), antipain (antipain), pepstatin (pepstatin), leupeptin (leupeptin), EGTA, PMSF (phenylmethylsulfonyl chloride), TLCK (sulfonyl lysine chloromethyl ketone), and the like can be used. The obtained body sample is further concentrated as necessary, and the detection sensitivity of amyloid spheres can be improved.

The detection of amyloid spheres in a biological sample using the anti-amyloid sphere-specific antibody can be performed by an immunological assay known per se. Specifically, for example, sandwich method, competition method (competition method), immunoassay method (immunoassay), nephelometry (nephelometry), and the like can be used. In the sandwich method, the amount of amyloid protein spheres in a biological sample can be measured by contacting the biological sample with the immobilized anti-amyloid sphere-specific antibody of the present invention, further reacting the antibody with the labeled anti-amyloid sphere-specific antibody, and then measuring a signal of the label bound to the solid phase. When the amount of amyloid spheres in a biological sample is measured by such an immunological measurement method, it is preferable to calculate the amount by using a calibration curve prepared using a standard solution containing known amounts of amyloid spheres. Specifically, experimental experiments can be performed according to experimental experiments in chemistry 11 "エンザイムイムノアツセイ" (same people in Beijing chemistry by Tijssen P., imperial science, imperial sciences, etc.), "Antibodies: a Laboratory Manual (Ed Harlow et al, Cold spring harbor LABORATORY (1988)) and the like, appropriately selected and combined. In addition, reagents for the detection of Alzheimer's disease individuals comprising anti-amyloid sphere-specific antibodies for use in these assays are also encompassed by the present invention.

Examples

The present invention will be described below with reference to examples, but the present invention is not limited to these examples. In the following examples and the present specification, "PBS" means "Phosphate buffer salt (Phosphate Buffered Saline)", "TTBS" means "Tween/Tris buffer salt (Tween-Tris Buffered Saline)", and "HRP" means "horseradish peroxidase (horserahdiepoxidase)".

Example 1: preparation of amyloid spheroid containing liquid

(1) Production of beta 40 amyloid protein (SEQ ID NO: 1) resin

342mg of Fmoc-Val resin (resin having an amine content of 0.73 mmol/g) was placed in an A433 type automatic peptide synthesizer manufactured by PerkinElmer Applied Biosystems, and supplied with Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asn Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Gln (trt) -OH, Fmoc-His (trt) -OH, Fmoc-Val-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-His trt) -OH, Fmoc-Arg (Pmc) -OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH, and HBTU (2- (1H-benzotriazol-1 yl) -1, 3, 3-tetramethyluronium hexafluorophosphate) was used as a condensing agent and sequentially condensed to obtain 1.515g of a side chain-protected β 40 amyloid resin.

(2) Trifluoroacetic acid treatment

304mg of the side chain-protected β 40 amyloid resin obtained in the above (1) was taken, and 0.75ml of phenol, 0.5ml of thioanisole, 8.25ml of trifluoroacetic acid, 0.25ml of ethane dithiol, and 0.5ml of distilled water were added thereto, and the mixture was cooled with ice for 5 minutes, followed by reaction at room temperature for 1.5 hours. After completion of the reaction, 200ml of ice-cooled diethyl ether was added to precipitate the peptide. The whole content was filtered through a glass filter, washed with cold diethyl ether, and then subjected to extraction treatment with 0.1% trifluoroacetic acid (about 200ml) containing 35% acetonitrile to obtain 191mg of a crude peptide represented by H-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Val-Val-OH.

(3) Purification of peptides

This crude peptide was dissolved in 0.1% trifluoroacetic acid (40ml) containing 35% acetonitrile, and purified by HPLC using a reverse phase column (inner diameter: 2cm, length: 25cm) having ODS (octadecylsilane) bound to silica. Elution was performed by ramping the acetonitrile concentration from 22% to 42% in 0.1% trifluoroacetic acid over 20 minutes. The obtained amount of the purified product was 35 mg. The structure of the substance was confirmed by MALDI-TOF mass analysis. Relative to the measured value [ M + H]+4330.99, calculated value (C)₁₉₄H₂₉₅N₅₃O₅₈S₁+ H) is 4330.89. In addition, as for the β 42 amyloid, both β 42 amyloid synthesized/produced according to the above method and β 42 amyloid purchased from Bachem corporation were used in the following experiment.

(4) Preparation of amyloid spheroid containing liquid

10nmol of the β 40 amyloid purified in the above (3) was placed in 1.5ml Eppendorftube, and 500. mu.l of ultrapure water and 500. mu.l of Dulbecco's phosphate buffer (-) (manufactured by Nissui Co., Ltd.; hereinafter also referred to as PBS (-)) were sequentially added thereto to completely dissolve the β amyloid. An Eppendorf tube containing the aqueous solution of amyloid β was attached to Duckrotor (manufactured by TAITEC, rotor: RT50), and rotated at 37 ℃ and 35rpm for 7 days to prepare an amyloid sphere 40. Amyloid spheres 42 were prepared by rotating amyloid β 42 (purified amyloid β 42 in (3) above or Bachem) for about 10 hours according to the method described above.

Example 2: preparation of hamster monoclonal anti-amyloid spheroid specific antibody

Amyloid spheres 42 prepared in PBS and an equal amount of Freund's complete adjuvant (WAKO) were mixed and emulsified, and then the immune system was performed subcutaneously in the dorsal area of the Immunity hamster with 0.2ml (16. mu.g/0.2 ml/hamster). Every 2 weeks, the cells were immunized in the same manner with amyloid spheres emulsified in Freund's incomplete adjuvant (Sigma-Aldrich). After 5 immunizations, plasma was prepared from carotid blood draw. The plasma was serially diluted with a 1% bovine serum albumin (BSA, fraction V; manufactured by Sigma-Aldrich Co.) solution (in PBS (-), and the reactivity to amyloid spheres by the following amyloid sphere solid phase ELISA method was measured.

The subjects who had well-increased reactivity after 6-12 immunizations were finally boosted by intraperitoneal administration of 16. mu.g of amyloid spheres (in PBS (-)0.2 ml). Splenocytes were recovered after 3 days of boosting and were subjected to cell fusion with 1/2 mouse myeloma cells (SP2/0-Agl4), which is the number of splenocytes, by a usual method using polyethylene glycol 4000. The cells obtained by the fusion were suspended in a GIT medium (WAKO) containing 10% fetal bovine serum, 10% BM conditioned H-1 (manufactured by Roche Diagnostics) and HAT (manufactured by Sigma-Aldrich), and the suspension was incubated at 5X 10 cells per well⁴Each myeloma cell was seeded in a 96-well plate (manufactured by FALCON) in a volume of 0.1ml of the culture medium. The culture medium was added 3 days later, the culture medium was changed 7 days later, and the supernatant was collected 2 to 3 days later. The supernatant was examined for anti-amyloid sphere-specific antibody by the following ELISA method, and the specific antibody-producing cells were expanded (expanded) in a 24-well plate (IWAKI). When cloning was performed by the limiting dilution method, hybridomas were seeded in 96-well plates at 200. mu.l of culture medium per well and cultured up to 0.3cell/well with half of the culture medium replaced every week.

The antibodies obtained from the thus obtained hamster monoclonal antibody-producing hybridomas H3-17-2-2 (hybridoma haASD1), H5-3-2-45 (hybridoma haASD2), H5-24-7, H5-47-10, and H4-3-5-4 are referred to as haASD1, haASD2, haASD3, haASD4, and haASD5, respectively.

Hybridoma haASD1 was deposited as FERM BP-10871 and hybridoma haASD2 was deposited as FERM BP-10872 in 2007 (19 years), day 7 and day 13 in japan independent administrative bodies of law, integrated research institute of industrial and technology, bioservation center of japan (central 6 of No. 1 of japan gasan-gun つくば a ghost imperial upon a mortem, No. 1 of fangassy).

The separation and purification of the antibodies obtained from hybridomas H3-17-2-2, H5-3-2-45, H5-24-7, H5-47-10, and H4-3-5-4 were carried out as follows. The Hybridoma was cultured in about 1L of CD Hybridoma medium (Invitrogen) for 1 week, and the culture supernatant was collected by centrifugation. After filtering the mixture through a 0.45 μm filter, 2ml of Protein-A agarose (Sepharose) equilibrated with PBS (-) was added thereto, and then an IgG antibody was isolated and purified in the same manner as in example 2(1) of WO 2006/016644.

EXAMPLE 3 analysis of antibody Properties

(1) Amyloid sphere solid phase ELISA method (confirmation of reactivity with amyloid spheres)

50. mu.l of amyloid spheres 42 diluted to 1. mu.g/ml in 1/2-concentration PBS (-) was added to a 96-well ELISA plate (MaxiSorp, Nunc) and left overnight at 4 ℃. After blocking the non-specific binding sites by adding a 1% bovine serum albumin (BSA, fraction V; manufactured by Sigma-Aldrich Co.) (in PBS (-)) solution at room temperature for 1 hour or more, the plates were washed with water. Mu.l of antiserum or hybridoma culture supernatant diluted with 1% bovine serum albumin solution (in PBS (-) was added and allowed to react at room temperature for 1 hour or more. The plate was washed 5 times with physiological saline containing 0.05% Tween20, and then peroxidase-labeled secondary antibody (anti-hamster IgG antibody (ROCKLAND Co.) diluted to 1. mu.g/ml) was added thereto in the same manner and allowed to react at room temperature for 1 hour. After washing 5 times, the substrate solution was added and allowed to develop color for a certain period of time, and the absorbance was measured with a microplate reader.

The results of a representative example of the hamster monoclonal antibody constructed in example 2 are shown in fig. 1. The antibody constructed in example 2 showed strong reactivity to amyloid spheres at low concentration.

(2) Dot blot analysis (confirmation of reactivity with amyloid spheres, amyloid beta fibers, amyloid beta monomers and amyloid precursor protein)

Beta 40 amyloid or 42 monomer protein dissolved in a solvent 1, 1, 1, 3, 3, 3, -hexafluoro-2-propanol (manufactured by BioRad corporation), amyloid sphere 40 or 42-containing solution prepared in example 1, A β fiber prepared from beta 40 amyloid, and commercially available amyloid precursor protein sAPP α (manufactured by Sigma) were blotted onto a nitrocellulose membrane (Schleicher & Schuell, 0.2 μ) using a Blotter (manufactured by BioRad corporation) at 5ng intervals, respectively, and the membrane was washed with PBS (-) and removed from the Blotter.

After blocking the blotted membrane with the above-mentioned proteins with 5% skim milk/0.05% TTBS for 1 hour, the membrane was dipped into rabbit polyclonal anti-amyloid sphere-specific antibody (rpASD1, rpASD2, rpASD3) (0.01. mu.g/mL), mouse monoclonal anti-amyloid sphere antibody (mASD3) (0.05. mu.g/mL), and antibody haASD1 (0.01. mu.g/mL) obtained in example 2, and reacted in a humidified chamber at 4 ℃ overnight. Then, the membrane was washed with 0.05% TTBS, and reacted with 0.05 to 1. mu.g/ml of anti-rabbit IgG, anti-mouse IgG or anti-hamster IgG conjugated with horseradish-derived peroxidase as a secondary antibody for 1 hour. Then, the sample was washed with 0.05% TTBS to remove the unreacted secondary antibody, immersed in SuperSignal West-Femto (manufactured by Pierce Co., Ltd.), incubated for 5 minutes, and then subjected to chemiluminescence signal detection with Image Analyzer "LAS-1000 plus" (manufactured by Fuji Photo Film Co., Ltd.) and Image data were obtained. As a control for the above antibody reactivity, 0.5. mu.g/mL of an anti-beta-amyloid antibody "6E 10" (manufactured by Senetek) and 0.04. mu.g/mL of an anti-APP N-terminal antibody "22C 11" (manufactured by Chemicon) were used as the primary antibody (primary antibody).

The results are shown in FIG. 2.

In fig. 2, a β 1-40 and a β 1-42 show the points of the monomer, the amyloid sphere 40-containing liquid prepared in example 1 and its 100kDa ultrafiltration membrane retentate fraction, and the amyloid sphere 42-containing liquid and its 100kDa ultrafiltration membrane retentate fraction, respectively; fibril denotes the point of A β fiber prepared from A β 1-40; in addition, sAPP α represents the spot of a commercially available amyloid precursor protein (manufactured by Sigma). As can be seen from the figure, the commercially available anti- β amyloid antibody "6E 10" reacted with all of the amyloid spheres 40, amyloid spheres 42, monomers, fibers, and sAPP α proteins prepared in example 1, whereas the hamster monoclonal antibody (haASD1) constructed in example 2 reacted with the amyloid spheres 40 and amyloid spheres 42 with high selectivity, but showed no reactivity with the amyloid precursor protein sAPP α.

(3) Dissociation constant determination

Amyloid spheres (42ASPD), amyloid beta monomers (42 Abeta, 40 Abeta), and Abeta fibers (fibril) prepared from 40 Abeta were coupled to a CM5 sensor chip of BIACore3000 (manufactured by BIAcore) in 50mM acetate buffer at 10. mu.g/ml. The binding rate constant and dissociation rate constant were determined using an antibody solution obtained by serially diluting 2-fold from the highest concentration of 100nM with 10mM HEPES, pH7.4, 0.15M NaC1, 3mM EDTA, and 0.005% Surfactant P20. The dissociation constant was calculated by the following equation using these constants.

Dissociation constant ═ dissociation rate constant/association rate constant

Table 1 shows dissociation constants (Kd) of mouse monoclonal anti-amyloid sphere antibody, hamster monoclonal anti-amyloid sphere specific antibody, and commercially available antibody (6E10) with respect to amyloid spheres.

The hamster monoclonal anti-amyloid spheroid specific antibody has a strong affinity for ASPD (Kd 10) as well as the mouse monoclonal anti-amyloid spheroid antibody^-11～10^-9M) and shows a higher selectivity than beta amyloid monomers or fibers (1 to 2 steps).

[ Table 1] dissociation constants of various anti-amyloid spheroid antibodies

Example 4: determination of epitopes of anti-amyloid-spheroid antibodies

(1) Antigenic determinants (epitopes) of anti-amyloid spheroid antibodies

To determine the epitope of an anti-amyloid spheroid antibody, a partial sequence of amyloid β monomer protein was chemically synthesized in order of 5 residues at a time from the N-terminus to obtain 38 partial sequence peptides each including amyloid β 5 residues (hereinafter, also abbreviated as a β in this example, and referred to as a β 1-5, a β 2-6, a β 3-7, a β 38-42 from the N-terminus). A.beta.was refined by HPLC to a single peak and then each was lyophilized quantitatively and stored at-20 ℃ until use.

The respective A.beta.s are dissolved in sterilized 0.5 XPBS (-) to prepare an A.beta. -antibody mixture solution such that the molar ratio of each A.beta.to each anti-amyloid spheroid antibody purified as IgG is 100 to 100 ten thousand times. Each a β -dilution was added to the amyloid sphere 40 solid phase plate prepared in example 3(1), shaken overnight at 4 ℃, washed with 0.01% Tween 20-PBS (-) solution, and then a peroxidase-bound secondary antibody (anti-rabbit antibody in the case of polyclonal antibody, anti-mouse antibody or anti-hamster antibody in the case of monoclonal antibody) diluted to 1 ten-thousandth of 1 was added thereto, and shaken for 1 hour. This was washed with a 0.01% Tween 20-PBS (-) solution, and developed using TMB Substrate Kit (manufactured by PIERCE). After the color development was stopped, the absorbance at 450nm was measured with a microplate reader (Benchmark; BioRad Co., Ltd.) to obtain the result.

The results are shown in FIG. 3. It is clear from the figure that haASD2 and 82E1 (manufactured by IBL corporation, Immunobiological research institute) which is a commercially available antibody are strongly competitively inhibited by the N-terminal peptide (A.beta.1-5) of the beta-amyloid monomeric protein. On the other hand, haASD1 and haASD3 were not competitively inhibited by the amyloid β 5 residue partial sequence peptide (even if the a β mixing ratio was increased to 100 ten thousand times in terms of molar ratio relative to the antibody). This result clearly suggests that the anti-amyloid sphere-specific antibody of the present invention is an antibody that recognizes a steric-structure-specific epitope of ASPD, unlike epitopes recognized by conventionally known antibodies.

Example 5: evaluation of neutralizing Activity of cytotoxicity of amyloid spheres

(1) Neutralization of amyloid spheroid toxicity by monoclonal anti-amyloid spheroid antibodies

The neutralizing activity of amyloid spheroid toxicity was evaluated using each monoclonal anti-amyloid spheroid antibody obtained in example 2. As an evaluation method, primary culture of nerve cells using rat hippocampus was performed. Primary culture of Hippocampus was prepared basically in the same manner as in the case of the forebrain base described in example 5 of WO2006/016644, except that the culture density was 1.0X 10⁵cells/cm²And (6) sowing. Amyloid spheres 42 prepared according to the method described in example 1 were used. The experimental conditions were as follows.

Concentration of amyloid spheres 42: 1.25. mu.M

Time of exposure to amyloid spheres: 45 hours

Cell: rat hippocampal neurons

Detection by PI staining

Monoclonal anti-amyloid spheroid antibody haASD2 exerts its effect of neutralizing neurotoxicity of amyloid spheroids even in hippocampal primary cultured nerve cells. As shown in FIG. 4, haASD2 showed concentration (5-50. mu.g/ml) dependent neutralization activity on the neurotoxicity of amyloid spheres.

Example 6: reactivity to amyloid precursor protein

(1) Western instant blotting

Western blotting was carried out using brain extracts of Tg2576 mice (Science 1996 Oct 4; 274 (5284): 99-102) overexpressing human APP (hAPP) in the brain, which is a model animal of Alzheimer's disease, as a sample, to examine the reactivity of the ASPD antibody and 6E 10. Specifically, TBS (Tris buffer salt) soluble fractions of cerebral cortex and hippocampus of Tg2576 mice (15 months of age) were treated with NuPAGE LDS Sample buffer, and about 50. mu.g of the Sample was applied to NuPAGENOVEX Bis-Tris Gel (4-12%) and subjected to electrophoresis (200V). Then, the transfer (30V, 60 min) was carried out on a nitrocellulose membrane, and the transferred membrane was blocked with TTBS (TBS containing 0.05% Tween 20) containing 5% skim milk (skim milk) (2 hours at room temperature). Then, the reaction mixture was reacted with each antibody (1. mu.g/ml) diluted with the same solution (overnight at 4 ℃), washed, and then detected with HRP-labeled secondary antibody (Superfemto).

As a result, in the Tg2576 mouse sample, 6E10 reacted with a β monomer and hAPP (bands indicated as a β monomer and APP in fig. 5), and the mouse ASPD monoclonal antibodies described in WO2006/016644 (mASD1, mASD2, and mASD3) did not react with a β monomer, but reacted with hAPP (bands indicated by arrows in fig. 6). On the other hand, the hamster ASPD monoclonal antibody of the invention did not show reactivity to a β monomer and hAPP as seen in 6E10 and mouse antibodies (fig. 7).

(2) Competitive ELISA

In the ELISA plate immobilized with ASPD, competition ELISA using each ASPD antibody and 6E10 was performed. Each antibody was previously reacted with ASPD or sAPP α (Sigma) in a separate plate (1 hour at room temperature) and added to ASPD-immobilized plates (blocked with 1% BSA). Then, the reaction was carried out at room temperature for 1 hour, and after washing, detection was carried out using an HRP-labeled secondary antibody.

As a result, as shown in Table 1, the mouse ASPD monoclonal antibody described in 6E10 and WO2006/016644 was directed not only to ASPD (IC)₅₀2.1 to 13nM) and shows reactivity (IC) to sAPP α₅₀9.6 to 33.8 nM). On the other hand, hamster antibodies showed reactivity to ASPD (IC)₅₀3.2-6.4 nM), but showed no significant reactivity to sAPP α (IC)₅₀Greater than or equal to 100 nM). From the above results, it is clear that the antibody of the present invention specifically reacts with amyloid spheres and does not significantly react with APPThe antibody thus produced is an excellent pharmaceutical product with less side effects and higher safety.

[ Table 2]

Example 7: test on organ specificity

The reactivity of the antibodies to normal human tissue was evaluated.

In the immunostaining using a mouse antibody, a frozen section of human tissue was fixed with acetone and then reacted with a Peroxidase blocking solution in an Envision kit of DAKO corporation for 5 minutes. After reacting with a protein blocking solution containing 0.5% casein, 1% bovine serum albumin, 1.5% normal goat serum, 2% normal human immunoglobulin, and 1mg/mL of heat-denatured human immunoglobulin for 20 minutes, an antibody diluted to a concentration of 2 or 10mg/mL with PBS containing 1% bovine serum albumin was added and allowed to react at room temperature for 1 hour. The resulting mixture was reacted with a Peroxidase labeled polymer in an Envision kit of DAKO corporation for 30 minutes, and then a DAB solution in an Envision kit of DAKO corporation was added thereto and reacted for 8 minutes. After all the above steps are completed, the specimen is washed with PBS before moving to the next step. After the immunostaining was completed, the specimens were washed with tap water and counterstained with hematoxylin.

In immunostaining with hamster antibodies, endogenous peroxidase was inactivated by adding glucose oxidase (2U/mL)/glucose (10mM) and sodium azide (1mM) to acetone-fixed frozen sections at 35 ℃ for 1 hour. Blocking with avidin solution at room temperature for 15 minutes, followed by blocking with biotin solution at room temperature for 15 minutes, and then reacting with a protein blocking solution containing 0.5% casein, 1% bovine serum albumin, 1.5% normal goat serum, 5% human immunoglobulin, 1mg/mL heat-modified human immunoglobulin at room temperature for 20 minutes. Hamster antibodies diluted to 2 or 10mg/mL with PBS containing 1% bovine serum albumin were added and allowed to react at room temperature for 1 hour, and then biotinylated goat anti-hamster IgG (H + L) antibody was added and allowed to react at room temperature for 30 minutes. It was further reacted with ABC Elite reagent for 30 minutes and thereafter with DAB solution at room temperature for 4 minutes.

All mouse anti-ASPD antibodies and hamster anti-ASPD antibodies tested stained senile plaque-like structures in frozen sections of alzheimer's patient brains, i.e. showed the presence of substances recognizing these antibodies in the brain brains of alzheimer's patients.

The test is carried out on normal human cerebellum, spinal cord, peripheral nerve, heart, liver and kidney, and as a result, a plurality of mouse anti-amyloid spheroid antibodies stain protein-like substances in or around blood vessels, nerve nets and nerve cell nuclei of normal brain tissues, smooth muscles, myofibroblasts, macrophages, Kupffer cells and the like.

Some hamster anti-amyloid spheroid antibodies stained skeletal muscle and cardiac myocytes, but were less reactive than mouse antibodies to normal human tissues. haASD1 and haASD2 hardly stained normal human tissues, and haASD2 in particular, showed no adhesion in each normal tissue. The results are shown in FIG. 8 and Table 3.

[ Table 3] comparison of confirmed immunopositive reactions in human tissue groups

	mASD1	mASD3	haASD1	haASD2
					Alzheimer's disease (age spots, neuron fiber changes)	+++	+++	+++	+++
Cerebellum (purkinje cell, glial cell)	+	±	±	-
					Heart and heart	+	-	±	-
Kidney (A)	+	+	-	-
					Liver disease	++	±	-	-
Peripheral nerve	+ + + (the same applies to Xuewang cells)	±	-	-
					Spinal cord	++	++	+	-
Blood vessel (endothelium, intravascular protein)	++	-	-	+ (blood vessels within the choroid plexus)

From the above results, it was found that the hamster monoclonal anti-amyloid spheroid antibody of the present invention is a specific antibody for the brain of alzheimer's disease with low cross-reactivity to human normal tissues, as compared with the mouse monoclonal anti-amyloid spheroid antibody known in the past. From this, it is assumed that therapeutic antibodies based on the specificity of these hamster antibodies can be used as therapeutic agents for alzheimer's disease with little side effects that do not affect tissues other than the target organ.

Example 8: examination of antibody specificity by immunoelectron microscopy

In a physiological solvent environment, ASPD or fibrillar amyloid β associates were reacted with 10 μ g/ml hamster antibody haASD 1. The reaction was carried out directly on a 1.5ml tube or a carbon evaporated Formvar grid. Then, the resulting mixture was reacted with a second antibody to which a 6nm gold colloid was bonded, and after negative staining with uranium acetate, the antibody was observed with an electron microscope.

As shown in fig. 9, the hamster antibody did not react with the fibrillar aggregates, but bound to the amyloid spheres.

Example 9: immunohistochemical analysis

Immunohistochemical analysis was carried out according to a prescribed method using frozen sections of optiumcut tempturecellular embedded frozen brain or sections 10 μm thick of paraffin-embedded formalin-fixed brain, which were derived from 10 Alzheimer's disease individuals (age 80.4. + -. 9.2 years, brain weight 964. + -.82 g, duration of disease 10.1. + -. 5.5 years) and 7 healthy individuals as controls (age 71.3. + -. 15.2 years, brain weight 1226. + -.96 g). As antibodies recognizing amyloid spheres, mouse monoclonal antibody mASD3, hamster monoclonal antibody haASD1, and rabbit polyclonal antibody rpASD2 were used. Further, as the anti- β -amyloid antibody, a commercially available anti- β -amyloid antibody "IBL 18582" (manufactured by IBL Co.) which recognizes the C-terminal end of β -amyloid (A β 1-42) and a commercially available anti- β -amyloid monoclonal antibody "6F/3D" (manufactured by DAKO Co.) which recognizes A β 8-17 were used.

As a result, all antibodies recognizing amyloid spheres (rpASD1, rpASD2, rpASD3, mASD3, haASD1) significantly stained plaques located in the frontal cortex, temporal cortex, and hippocampus (senile plaques, diffuse senile plaques) of the frozen sections of alzheimer's disease brain. This shows that amyloid spheroid-like structures are present in situ (in situ) within the plaque. In addition, these antibodies stained plaques of formalin-fixed paraffin sections of brain with or without microwave treatment or formic acid treatment, except haASD 1. In addition, in normal control brains of the same age, structures other than individual plaques were not stained. On the other hand, a commercially available anti- β amyloid antibody "IBL 18582" recognizing the C-terminal end of β amyloid (A β 1-42) and a commercially available anti- β amyloid monoclonal antibody "6F/3D" recognizing A β 8-17 hardly stained plaques in a frozen section or a paraffin section of Alzheimer's disease brain without microwave oven treatment or formic acid treatment. From the above results, it was found that an antibody recognizing an amyloid sphere specifically recognizes the structure of an amyloid sphere, particularly haASD1, which is an antibody recognizing a three-dimensional structure more finely. This result is consistent with the result that the amyloid sphere antibody does not react with the fibrous aggregate and binds to the amyloid sphere in the test of antibody specificity by immunoelectron microscopy in example 8.

Example 10: obtaining and analysis of humanized antibodies

(1) Obtaining of humanized antibody

RNA was obtained from the hamster monoclonal antibody-producing hybridoma haASD2 obtained in example 2 using RNeasy mini kit (cat.no.74106) manufactured by QIAGEN. Using this RNA as a template, cDNA was synthesized using 1st strand cDNA synthesis kit (Cat. No.27-9261-01) from GE Life Sciences. For the light chain variable region, the cDNA was amplified using primers haVK1 and hacK and Phusion High-Fidelity PCR Master Mix (Cat. No. F-531S) from Finnzymes and ligated with pCR-Blunt II-TOPO vector from Zero Blunt TOPO PCR Cloning Kit (Cat. No.450245) from Invitrogen. For the heavy chain variable region, the cDNA was amplified using primers haVHf and MHCG3 and AdvantageR-HF 2 PCR Kit (Cat. No.639123) manufactured by Clontech and ligated with pCR2.1-TOPO vector of TOPO-TA cloning Kit (Cat. No.450641) manufactured by Invitrogen. After introducing these into TOP10(Cat. No.404003) made by E.coli competent cell Invitrogen, the GATC Biotech company was requested to determine the DNA sequence by analyzing the nucleotide sequence of a clone containing an insert DNA (VH: about 730bp, VL: about 850bp) of the desired size.

[ Table 4] PCR primers for cloning hamster VL

Name (R)	Sequence (5 '→ 3')
		haVK1	ATGGCTTGGACTCCTGGC (Serial number 19)
haCK	GTCTTCACCCCATCATTGATAG (Serial number 20)

[ Table 5] PCR primers for cloning hamster VH

Name (R)	Sequence (5 '→ 3')
		haVHf	ATGGGGTTGGGGCTGCACTGGG (Serial number 21)
MHCG3	CAAGGGATAGACAGATGGGGC (Serial number 22)

The Complementarity Determining Regions (CDRs) in the variable regions of the light and heavy chains of hamster antibodies were determined according to the method of Kabat et al ("Sequences of Proteins of Immunological Interest", Kabat, E., et al, US Department of Health and Human Services, (1983)), and hamster antibody haASD2 was humanized according to the method of Winter described in Japanese patent No. 2912618. As a result, 2 kinds of humanized antibodies were obtained and namedThese are RHA/RLA (hereinafter sometimes referred to as huaSD2) and RHB/RLB.

An expression plasmid into which the heavy chain cDNA of the humanized antibody huASD2 was introduced was prepared and designated ASD2RHApG1D 200. An expression plasmid into which the light chain cDNA of the humanized antibody RHA/RLA was introduced was prepared and designated as ASD2RLApLN 100. The reception numbers of ASD2RHApG1D200 and ASD2RLApLN100 are FERM ABP-11040 (accession number: FERM BP-11040) and FERM ABP-11041 (accession number: FERM BP-11041) respectively, which were both deposited at the national institute of independent administrative Fairy, institute of Industrial science and technology, Biodepositary, 10.17.2008 (Central No.6 of Anmo 1 Anmo, 1, Mount, Shang, Japan, Kagawa つくば ( Kaisano. 305 & 8566)).

The humanized antibody huASD2 can be obtained by co-transfecting ASD2RHApG1D200 and ASD2RLApLN100 into a known animal cell (e.g., CHO, NS0, HEK293, COS, etc.) and expressing the cell. As an example, the transient expression of HEK293 was used, which was manufactured by Invitrogen FREESTYLE MAX 293 EXP SYSTEM (Cat. No. K9000-10).

The DNA and amino acid sequences of the heavy chain variable region of humanized antibody huASD2 are shown in seq id nos 4 and 5 of the sequence listing and fig. 10. The respective positions of the CDRs 1, 2, 3 are shown in fig. 10.

The DNA and amino acid sequences of the light chain variable region of humanized antibody huASD2 are shown in seq id nos 6 and 7 of the sequence listing and fig. 11. The respective positions of the CDRs 1, 2, 3 are shown in fig. 11.

The humanized antibody RHB/RLB can be obtained as follows, similarly to the humanized antibody huaSD 2: plasmids expressing the heavy chain and the light chain, respectively, were prepared, co-transfected into animal cells, and then expressed.

SEQ ID NO. 8 of the sequence Listing and FIG. 12 show the amino acid sequence of the heavy chain variable region of the humanized antibody RHB/RLB. The respective positions of the CDRs 1, 2, 3 are shown in fig. 12.

SEQ ID NO. 9 of the sequence Listing and FIG. 13 show the amino acid sequence of the light chain variable region of the humanized antibody RHB/RLB. The respective positions of the CDRs 1, 2, 3 are shown in fig. 13.

Furthermore, the humanized antibody RHC/RLC can be obtained by changing the humanized antibody huaSD 2. The amino acid sequence of the heavy chain variable region of the humanized antibody RHC/RLC was identical to the heavy chain variable region of the humanized antibody huaSD 2. The amino acid sequence of the light chain variable region of the humanized antibody RHC/RLC is shown as the sequence number 10 in the sequence table and figure 14.

(2) Analysis of humanized antibodies

FIG. 15 is a graph comparing the amino acid sequences of the heavy chain variable region and the light chain variable region of humanized antibodies huaSD2, RHB/RLB and RHC/RLC. In the figure, the heavy chain variable region sequence of humanized antibody huASD2 is denoted as ASD2RHA, the light chain variable region sequence is denoted as ASD2RLA, the heavy chain variable region sequence of humanized antibody RHB/RLB is denoted as ASD2RHB, the light chain variable region sequence is denoted as ASD2RLB, and the light chain variable region sequence of humanized antibody RHC/RLC is denoted as ASD2 RLC. The amino acid sequence of the heavy chain variable region of the humanized antibody RHC/RLC was identical to the amino acid sequence of the heavy chain variable region of the humanized antibody huaSD2 (ASD2RHA of FIG. 15), and thus omitted in FIG. 15.

In fig. 15, the humanized antibody RHB/RLB heavy chain variable region can be prepared by replacing amino acid positions 49 (G), 81 (L) and 100 (T) in the heavy chain variable region of humanized antibody huASD2 with A, V and R, respectively. In addition, the light chain variable region of humanized antibody RHB/RLB can be prepared by replacing amino acid numbers 48 (Y), 49 (L), 51 (K), 74 (a) and 79 (G) in the light chain variable region of humanized antibody huASD2 with F, F, F, T and a, respectively.

Among the amino acid residues shown in fig. 15, sequences other than the CDRs of the heavy chain variable region (ASD2RHA) and the light chain variable region (ASD2RLA) of the humanized antibody huASD2 were structurally important as judged by analysis of Medical research chain company (hereinafter, MRCT) according to the method of Winter described in japanese patent No. 2912618, and the properties of huASD2 were more effectively modified/changed by substituting them. Specifically, in the humanized antibody huASD2 and the hamster antibody haASD2, when there is a functional difference, it is expected that the difference can be reduced or eliminated by reverting one or several positions or all of them to the sequence of the original hamster antibody haASD2 (back mutation). As an example, the sequence RHB/RLB (SEQ ID NOS: 8 and 9, ASD2RHB and ASD2RLB of FIG. 15) can be cited. However, from the viewpoint of humanization for the purpose of reducing the immunogenicity to a human having a heterodominance antibody (heterologous leader antibody), the amino acid substitution described herein is not a preferred procedure, and is not necessarily carried out if the effect on the immunogenicity is not enhanced.

On the other hand, when the sequences of human antibodies AB021517(GenBank accession number) and AJ241418(GenBank accession number) for designing the variable region framework of huASD2 were compared with the original genomic sequences (each V segment, J segment), respectively, hypermutation of the light chain (seq id nos. 6 and 7) was observed. Hypermutations are mutations visible during antigen-specific maturation of antibodies, which occur in an immunological environment that reflects the genetic background of an individual. Therefore, if the genetic background is different, immunogenicity may be shown, which is considered to be a factor of the unexpected immunogenicity found in the fully human antibody Humira.

As a means for avoiding the occurrence of the mutation, it is considered to restore the amino acid residues having the hypermutation to a human common genomic sequence. Specifically, the 1-position, 2-position, or all of the 3 (amino acids 2 (S), 8 (S), and 51 (K) of seq id No. 7) hypermutated amino acid residues in the huASD2 light chain variable region (ASD2RLA of fig. 15) were replaced with the respective genomic sequences A, A and R (ASD 2RLC of fig. 15, seq id No. 10), within the range of tolerable influence on antibody properties.

Example 11: analysis of humanized antibody Properties

(1) Dissociation constant determination

In the same order as in example 3(3), 42ASPD, 40A β (a β monomer) and a β fiber (42fibril) prepared from 42A β were coupled to a CM5 sensor chip, and dissociation constants of hamster antibody haASD2, humanized antibody huASD2 and anti-a β antibody 3D6(US20030165496a1) for the above proteins were determined. The anti- Α β antibody 3D6 was prepared by a known method to a synthetic gene corresponding to the mouse antibody light chain variable region 3d6vl.aa and its heavy chain variable region 3d6vh.aa described in US20030165496a1, and based on the information of this patent, was linked to the light chain constant region and the heavy chain constant region of a known mouse antibody, respectively, to produce a mouse IgG2b/κ molecule.

The results are shown in Table 6. Humanized antibody huASD2 showed strong affinity for ASPD (Kd 1.8X 10)^-10M) and has an affinity about 580 times higher than that of the β amyloid monomer and about 6.4 times higher than that of the amyloid fiber. On the other hand, the affinity of the anti-a β antibody 3D6 for ASPD was about 34 times higher than that of β amyloid monomer and about 2.0 times higher than that of amyloid fiber. That is, we obtained the humanized antibody huASD2 at this time with higher selectivity for ASPD than the well-known a β antibody 3D 6. That is, the humanized antibody huASD2 of the present invention has low binding to a β 40 deposited in the cerebral blood vessels, and therefore, it is expected that the side effect of bleeding from the cerebral micro-vessels is small.

[ Table 6] dissociation constants of antibodies

(2) Determination of the epitope of humanized antibody huASD2

Using the same method as in example 4, it was investigated to which a β 5 residue peptide humanized antibody huASD2 bound. From the results, it was found that the humanized antibody huASD2 was strongly competitively inhibited by the peptide (a β 1-5) at the N-terminal end of the β amyloid monomer protein, similarly to the hamster antibody haASD 2.

Example 12: evaluation of neutralizing Activity of cytotoxicity of humanized antibody RHA/RLA

Fetal mice were removed from 17-day-old pregnant rats (SD, Charles River, japan), and then the hippocampus was enucleated from the brain thereof. Hippocampal primary cultured neural cells were prepared in the same manner as described in example 5 of WO 2006/016644. Specifically, hippocampal neurons were cultured in a neuronal cell culture medium (SUMILON) (37 ℃, 5% CO)₂) And 5 days. For ASPD, ASPD obtained according to a known method (proc. natl. acad. sci. usa, 100, 6370-. DF-ASPD (0.35. mu.M) was incubated with haASD2 or huASD2(7.5, 25 and 75. mu.g/ml) at room temperature for 2 hours, then added directly to the culture broth and cultured for 24 and 45 hours. As a control, PBS (-) without antibody was used. Thereafter, neuronal cell death was detected by celllife ELISA (Rosch) and Pyridine Iodide (PI) staining.

The results of evaluating the neutralizing activity of haASD2 and huASD2 against DF-ASPD-induced neuronal cell death by using the cell death ELISA, a detection system for apoptosis, are shown in fig. 16. In fig. 16, F12 is a control without DF-ASPD. By adding DF-ASPD, OD (405-492nm) values increased, confirming the presence of apoptosis induction (DF-ASPD (0.35. mu.M), PBS (-) column). In contrast, the humanized antibody huASD 2-added group of hamster antibody haASD2 showed a significant decrease in OD value (columns of DF-ASPD (0.35 μ M), huASD2(7.5, 25 and 75)), and a high inhibitory effect on DF-ASPD-induced neuronal cell death.

Next, the results of evaluating the neutralizing activity of haASD2 and huaasd 2 antibodies (25 μ g/ml) against DF-ASPD-induced neuronal cell death by detecting cell death using PI staining are shown in fig. 17. Cell death was detected by measuring the number of PI positive cells per field. In the same manner as in fig. 16, F12 is a control containing no DF-ASPD, and PBS (-) is a control containing no antibody. PI-positive cells were significantly increased by the addition of DF-ASPD (0.35 μ M), confirming the presence of induction of cell death (DF-ASPD (0.35 μ M), PBS (-) column). In contrast, haASD2 and huASD2 showed significant inhibitory effects on the number of PI-positive cells, and were highly effective in inhibiting neuronal cell death caused by DF-ASPD-induced cell death.

The antibody of the present invention has low reactivity to amyloid precursor protein, higher reactivity to amyloid spheres than to beta-amyloid fibrils or beta-amyloid monomer protein, and has activity of inhibiting induction of neuronal cell death by amyloid spheres. Since amyloid spheres induce neuronal cell death at a concentration equivalent to that of β amyloid present in the brain of alzheimer's disease patients, amyloid spheres can be used as a therapeutic or prophylactic agent for alzheimer's disease by obtaining (1) an antibody having an activity of inhibiting formation of amyloid spheres or (2) an antibody having an activity of inhibiting induction of neuronal cell death by amyloid spheres. In addition, if (3) an antibody having higher reactivity to amyloid spheres than to amyloid beta monomer protein or amyloid beta fibrils is obtained, it can also be applied to the detection of alzheimer's disease individuals. The antibody of the present invention has low reactivity to amyloid precursor protein and high brain specificity, and therefore can be a therapeutic agent for alzheimer's disease with higher safety than the conventionally known anti-amyloid spheroid antibodies.

Sequence listing

<110> Mitsubishi chemical corporation

<110> Mitsubishi pharmaceutical corporation

<120> antibodies and uses thereof

<130>A81495A

<160>22

<210>1

<211>40

<212>PRT

<213>Human

<400>1

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

1 5 10 15

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

20 25 30

Gly Leu Met Val Gly Gly Val Val

35 40

<210>2

<211>42

<212>PRT

<213>Human

<400>2

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

1 5 10 15

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

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

<210>3

<211>43

<212>PRT

<213>Human

<400>3

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

1 5 10 15

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

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala Thr

35 40

<210>4

<211>360

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：humanized antibody

<220>

<221>CDS

<222>(1)..(360)

<223>

<400>4

gaa gtg cag ctg gtc gag tct ggc ggc gga ctc gtg aag cct ggc ggc 48

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

1 5 10 15

tcc ctg cgg ctg tcc tgc gcc gcc tcc ggc ttt acc ttc tcc gac tac 96

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

ttc atg tcc tgg gtg cgg cag gct cct ggc aag ggc ctg gaa tgg gtc 144

Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

ggg ggg atc gag atc aag tcc tacttc tac gcc acc tac tac ttc ggc 192

Gly Gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly

50 55 60

tcc gtg aag ggc cgg ttc acc atc tcc cgg gac gac tcc aag aac acc 240

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

ctg tac ctg cag atg aac tcc ctg aaa acc gag gac acc gcc gtg tac 288

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

tac tgc acc acc aac cgg gaa gtg ggg ggc ctg gac aac tgg ggc cag 336

Tyr Cys Thr Thr Asn Arg Glu Val Gly Gly Leu Asp Asn Trp Gly Gln

100 105 110

ggc acc ctg gtg acc gtg tcc tcc 360

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>5

<211>120

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：humanized antibody

<400>5

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asn Arg Glu Val Gly Gly Leu Asp Asn Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>6

<211>351

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：humanized antibody

<220>

<221>CDS

<222>(1)..(351)

<223>

<400>6

cag tcc gtg ctg acc cag cct tcc tcc ctg tcc gcc tcc cct ggc gcc 48

Gln Ser Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala

1 5 10 15

tcc gcc tcc ctg acc tgc acc ctg cgg tcc ggc atc tcc gtg ggc ggc 96

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly

20 25 30

aag aac atc tac tgg tat cag cag aag cct ggc tcc cct cct cag tac 144

Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr

35 40 45

ctg ctg aag tac tcc tcc tac tcc aac aag cag ctg gga cct ggc gtg 192

Leu Leu Lys Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val

50 55 60

cct tcc cgg ttc tcc ggc tcc aag gac gcc agc gcc aac gcc ggc atc 240

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile

65 70 75 80

ctg ctg atc tct gga ctg cag agc gag gac gag gcc gac tac tac tgc 288

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

tcc atc cac gag tcc aac gcc tac gtg ttt ggc ggc gga aca aag ctg 336

Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu

100 105 110

aca gtc ctg ggc cgg 351

Thr Val Leu Gly Arg

115

<210>7

<211>117

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：humanized antibody

<400>7

Gln Ser Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala

1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly

20 25 30

Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr

35 40 45

Leu Leu Lys Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile

65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu

100 105 110

Thr Val Leu Gly Arg

115

<210>8

<211>120

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：humanized antibody

<400>8

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asn Arg Glu Val Gly Gly Leu Asp Asn Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>9

<211>116

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：humanized antibody

<400>9

Gln Ser Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala

1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly

20 25 30

Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Phe

35 40 45

Phe Leu Phe Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Thr Ser Ala Asn Ala Ala Ile

65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu

100 105 110

Thr Val Leu Gly

115

<210>10

<211>116

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：humanized antibody

<400>l0

Gln Ala Val Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Ala

1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly

20 25 30

Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr

35 40 45

Leu Leu Arg Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile

65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu

100 105 110

Thr Val Leu Gly

115

<210>1l

<211>5

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<400>11

Asp Tyr Phe Met Ser

1 5

<210>12

<211>19

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<400>12

GlyIle Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly Ser Val Lys Gly

1 5 10 15

<210>13

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<400>13

Asn Arg Glu Val Gly Gly Leu Asp Asn

1 5

<210>14

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<400>14

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

1 5 10

<210>15

<211>11

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<400>15

Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro

1 5 10

<210>16

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<400>16

Ser Ile His Glu Ser Asn Ala Tyr Val

1 5

<210>17

<211>120

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<220>

<222>(49)

<223>Gly or Ala

<220>

<222>(81)

<223>Leu or Val

<220>

<222>(100)

<223>Thr or Arg

<400>17

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Phe Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Xaa Gly Ile Glu Ile Lys Ser Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Xaa Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Xaa Asn Arg Glu Val Gly Gly Leu Asp Asn Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>18

<211>116

<212>PRT

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：recombinant antibody

<220>

<222>(2)

<223>Ser or Ala

<220>

<222>(8)

<223>Ser or Ala

<220>

<222>(48)

<223>Tyr or Phe

<220>

<222>(49)

<223>Leu or Phe

<220>

<222>(51)

<223>Lys，Phe or Arg

<220>

<222>(74)

<223>Ala or Thr

<220>

<222>(79)

<223>Gly or Ala

<400>18

Gln Xaa Val Leu Thr Gln Pro Xaa Ser Leu Ser Ala Ser Pro Gly Ala

1 5 10 15

Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Ser Val Gly Gly

20 25 30

Lys Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Xaa

35 40 45

Xaa Leu Xaa Tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Xaa Ser Ala Asn Ala Xaa Ile

65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

Ser Ile His Glu Ser Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu

100 105 110

Thr Val Leu Gly

115

<210>19

<211>18

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：Synthetic DNA

<400>19

atggcttgga ctcctggc 18

<210>20

<211>22

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：Synthetic DNA

<400>20

gtcttcaccc catcattgat ag 22

<210>21

<211>22

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：Synthetic DNA

<400>21

atggggttgg ggctgcactg gg 22

<210>22

<211>21

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：Synthetic DNA

<400>22

caagggatag acagatgggg c 21

Claims

1. A monoclonal antibody which has higher reactivity to amyloid spheres than to amyloid precursor protein and has any one of 1 or more of the following characteristics:

(iii) has the activity of inhibiting the induction of nerve cell death by amyloid spheroids,

the antibody is produced by the hybridoma having a deposit number FERM BP-10871 or FERM BP-10872.

2. A humanized antibody or a fragment thereof obtained by humanizing a hamster monoclonal antibody produced from a hybridoma deposited with the FERM BP-10871 or FERM BP-10872, which comprises a humanized heavy chain and a humanized light chain,

the humanized heavy chain comprises 3 heavy chain complementarity determining region CDRs derived from a hamster monoclonal antibody produced by the hybridoma having the deposit number FERM BP-10872, and a heavy chain variable region framework sequence derived from a human immunoglobulin heavy chain;

the humanized light chain comprises 3 light chain complementarity determining region CDRs derived from a hamster monoclonal antibody produced by the hybridoma having the deposit number FERM BP-10872, and a light chain variable region framework sequence derived from a human immunoglobulin light chain;

and 3 heavy chain complementarity determining region CDRs each having an amino acid sequence of: heavy chain CDR 1: asp Tyr Phe Met Ser, serial No. 11; heavy chain CDR 2: gly Ile Glu Ile LysSer Tyr Phe Tyr Ala Thr Tyr Tyr Phe Gly Ser Val Lys Gly, SEQ ID NO. 12; and heavy chain CDR 3: asn Arg Glu Val Gly Gly Leu Asp Asn, serial No. 13;

the 3 light chain complementarity determining region CDRs have the following amino acid sequences: light chain CDR 1: ThrLeu Arg Ser Gly Ile Ser Val Gly Gly Lys Asn Ile Tyr, sequence No. 14; light chain CDR 2: tyr Ser Ser Tyr Ser Asn Lys Gln Leu Gly Pro, serial number 15; and light chain CDR 3: ser Ile His Glu Ser Asn Ala Tyr Val, serial number 16.

3. A humanized antibody or a fragment thereof obtained by humanizing a hamster monoclonal antibody produced from a hybridoma having a deposit number of FERM BP-10871 or FERM BP-10872, which comprises a humanized heavy chain variable region and a light chain variable region;

the humanized heavy chain variable region comprises the following amino acid sequence, namely the sequence No. 17:

the light chain variable region comprises the following amino acid sequence, namely the sequence number 18:

4. The humanized antibody or a fragment thereof according to claim 3, which has a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 5 and a light chain variable region having an amino acid sequence shown in SEQ ID NO. 7.

5. A screening method for a therapeutic and/or prophylactic agent for alzheimer's disease, which comprises: a test substance and the antibody according to any one of claims 1 to 4 are brought into contact with an amyloid sphere, and a candidate substance is selected using the binding property of the test substance to the amyloid sphere as an index.

6. A neuroprotective agent comprising the antibody according to any one of claims 1 to 4.

7. A reagent for detecting Alzheimer's disease, which comprises the antibody according to any one of claims 1 to 4.

8. A pharmaceutical product comprising the antibody according to any one of claims 1 to 4.

9. A therapeutic and/or prophylactic agent for Alzheimer's disease, which comprises the antibody according to any one of claims 1 to 4.

10. Hybridomas having a deposit number of FERM BP-10871 or FERM BP-10872.

11. A nucleic acid comprising a sequence encoding a heavy chain or a light chain of a humanized antibody according to any one of claims 2 to 4, or a fragment thereof.

12. An expression vector for expressing the humanized antibody or fragment of any one of claims 2 to 4 comprising a nucleotide sequence encoding said antibody or fragment.