JP2005104975A - Use of antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a more effective and safety preventive/remedy for Alzheimer's disease. <P>SOLUTION: The preventive/remedy for Alzheimer's disease and or the like which contains a monoclonal antibody reacting specifically with a partial peptide in the C-terminal side of beta-amyloid or its derivative. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a preventive / therapeutic agent or diagnostic agent for Alzheimer's disease and the like. More specifically, the present invention relates to a prophylactic / therapeutic agent or diagnostic agent for Alzheimer's disease and the like comprising a monoclonal antibody that specifically reacts with a partial peptide on the C-terminal side of β-amyloid (Aβ) or a derivative thereof.

  Senile dementia due to Alzheimer's disease has become a major social problem, and early establishment of a method for preventing and treating Alzheimer's disease is desired. As a characteristic lesion in the brain of Alzheimer's disease patients, excessive formation of senile plaque amyloid and neurofibrillary tangles are known, and among these, one of the main components of senile plaque is β-amyloid or its derivatives. is there.

β-amyloid is a peptide composed of about 40 to 43 amino acids, and is encoded in the vicinity of the transmembrane region of amyloid precursor protein (hereinafter referred to as APP). The amino acid sequence of each β-amyloid is as follows.
β-amyloid (1-38) = SEQ ID NO: 1
β-amyloid (1-39) = SEQ ID NO: 2
β-amyloid (1-40) = SEQ ID NO: 3
β-amyloid (1-41) = SEQ ID NO: 4
β-amyloid (1-42) = SEQ ID NO: 5
β-amyloid (1-43) = SEQ ID NO: 6

  More recently, among β-amyloids, β-amyloid (1-42) is deposited mainly in the cerebral parenchyma (senile plaques) mainly from the early stage, while β-amyloid (1-40) is deposited in the cerebral blood vessels. It is reported that it mainly deposits (amyloid angiopathy) [Non-patent document 1 (Arch. Biochem. Biophys., 301, 41-53, 1993)], β-amyloid (1 -42), β-amyloid (26-42), β-amyloid (26-43), β-amyloid (34-42), etc. It has been suggested to cause deposition of (1-40) [Non-Patent Document 2 (Biochemistry, 32, 4693-4697, 1993)]. Therefore, the deposition of β-amyloid (x-42) is considered to be the most important etiology in Alzheimer's disease. From such reports, β-amyloid (x-40) and β-amyloid (x-42) It is considered that the difference in the deposition pattern with Alzheimer's disease greatly contributes to Alzheimer's disease.

  For this reason, it is thought that it is important to separate and quantify β-amyloid (x-40) and β-amyloid (x-42) with high sensitivity, and the partial peptide on the C-terminal side of β-amyloid or a derivative thereof is considered to be important. An antibody that reacts specifically and a method for measuring β-amyloid having a C-terminal hydrophobic region using the antibody are reported in Patent Document 1 (WO 94/17197). The preventive and therapeutic effects of Alzheimer's disease are not known.

On the other hand, regarding the direct treatment of Alzheimer's disease with β-amyloid vaccine, for example, Non-Patent Document 3 (Nature (Nature), 400, 173-177, 1999), Non-Patent Document 4 (Nature Medicine) 9 (4), 448-452, 2003), etc., but in a preclinical study using human APP transgenic mice, etc., it was successful in reducing senile plaques and improving learning ability. Despite its promise as an effective treatment, it has led to unexpected serious side effects in recent phase II clinical trials. The main cause of serious side effects that caused deaths is thought to be the onset of encephalitis due to infiltration of CD4 ⁺ lymphocytes into the brain, which is not seen in normal Alzheimer's disease.

  As a result of immunotherapy, it has been confirmed in humans that senile plaques deposited in the brain are dramatically reduced and the cognitive ability is improved [Non-Patent Document 10 (Neuron, 38, 547- 555, 2003)], one option for avoiding side effects while taking advantage of this advantage is passive immunization by peripheral administration of anti-β-amyloid antibody, which has been studied at the animal level using monoclonal antibodies. [Non-patent document 5 (Science, 295, 2264-2267, 2002), Non-patent document 6 (Nature Neuroscience, 5, 452-457, 2002)]. However, in studies using cerebral amyloid angiopathy (CAA) model mice, administration of anti-N-terminal β-amyloid antibody has been reported to cause exacerbation of hemorrhage from cerebral blood vessels caused by CAA [Non-patent Document 7]. (Science, 298, 1379, 2002)].

In an experiment of passive immunization at the animal level using an antibody having an epitope at the N-terminus or middle part of β-amyloid, an increase in blood β-amyloid concentration has been reported [Non-patent document 5, Non-patent document Reference 6, Non-patent document 8 (Nature Medicine, 8, 1263-1269, 2002), and Non-patent document 9 (Proceedings of the National Academy of Sciences of the USA), 100, 2023-2028, 2003)], and there is no report of immunotherapy that specifically increased the blood concentration of highly aggregating β-amyloid (x-42).
WO 94/17197 Arch. Biochem. Biophys., 301, 41-53, 1993 Biochemistry, 32, 4693-4697, 1993 Nature, 400, 173-177, 1999 Nature Medicine, 9 (4), 448-452, 2003 Science, 295, 2264-2267, 2002 Nature Neuroscience, 5, 452-457, 2002 Science, 298, 1379, 2002 Nature Medicine, 8, 1263-1269, 2002 Proceedings of the National Academy of Sciences of the USA, 100, 2023-2028, 2003 Neuron, 38, 547-555, 2003

  It is pointed out that β-amyloid may be one of the causative agents of Alzheimer's disease, and despite the deep interest in β-amyloid, effective prevention and treatment methods have not been established. It is. As for the immunotherapy of Alzheimer's disease, an antibody that specifically reacts with a partial peptide at the N-terminal side, intermediate part of β-amyloid, or C-terminal part of β-amyloid (x-40), for example, cerebral blood vessels Considering the risk of causing inflammation of β-amyloid (x-40) (CAA) deposited on the wall and causing hemorrhage in the brain, it is always a sufficiently safe prevention and treatment method for Alzheimer's disease. I can't say that.

  As described above, there is an urgent need for a more effective and safe Alzheimer's disease prevention / treatment method.

  As a result of intensive studies to solve the above problems, the present inventors have subcutaneously introduced a monoclonal antibody BC-05a that specifically reacts with a partial peptide on the C-terminal side of β-amyloid into a model mouse of Alzheimer's disease. Surprisingly, the β-amyloid (x-42) -only blood was suppressed without increasing the concentration of β-amyloid (x-40) in the blood. As a result of further finding out based on these findings, the present invention has been completed.

That is, the present invention
(1) Alzheimer's disease comprising a monoclonal antibody that specifically reacts with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof and does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 8 , Preventive or therapeutic agent for mild cognitive impairment or brain amyloid angiopathy,
(2) The agent according to the above (1), which is a prophylactic / therapeutic agent for Alzheimer's disease,
(3) The agent according to (1), wherein the antibody is an antibody that does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 9.
(4) The agent according to (1) above, wherein the antibody is an antibody that recognizes a partial peptide having the amino acid sequence represented by SEQ ID NO: 9,
(5) β-amyloid is a peptide having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 (1 ) Agents described,
(6) The agent according to (1) above, wherein β-amyloid is a peptide having the amino acid sequence represented by SEQ ID NO: 5.
(7) A peptide having β-amyloid derivative having the second to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 5, and the third to 42nd amino acid sequence represented by SEQ ID NO: 5 The peptide according to (1), which is a peptide having an amino acid sequence, wherein the N-terminal glutamic acid is converted to pyroglutamic acid, or a peptide having the 4th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 5. Agent,
(8) The β-amyloid derivative is a peptide having an amino acid sequence lacking the first to tenth amino acid sequences from each amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 6, The agent according to (1) above, wherein glutamic acid is a peptide converted to pyroglutamic acid,
(9) The agent according to (1) above, wherein the partial peptide on the C-terminal side of β-amyloid or a derivative thereof is a partial peptide having an amino acid sequence from the 25th amino acid counting from the N-terminal amino acid of each β-amyloid,
(10) The agent according to (1) above, wherein the antibody is an antibody that does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 7.
(11) The agent according to (1) above, wherein the antibody is an antibody that recognizes β-amyloid (1-42) having the amino acid sequence represented by SEQ ID NO: 5.
(12) The agent according to (1) above, wherein the antibody is a monoclonal antibody BA-27a that can be produced from a hybridoma cell labeled with BA-27 (FERM BP-4139),
(13) The agent according to (1), wherein the antibody is a monoclonal antibody BC-05a that can be produced from a hybridoma cell labeled BC-05 (FERM BP-4457),
(14) The agent according to (1) above, wherein the antibody is an antibody that penetrates the blood-brain barrier,
(15) The agent according to (14), wherein the antibody is an antibody that can extract β-amyloid from the formed senile plaques,
(16) The agent according to (1) above, which is an inhibitor of aggregation or deposition of β-amyloid in the brain,
(17) The agent according to (1) above, which can specifically increase the blood concentration of the peptide having the amino acid sequence represented by SEQ ID NO: 5,
(18) The agent according to (1) above, wherein the antibody is an antibody that does not penetrate the brain-blood barrier,
(19) The agent according to (18), wherein the antibody is an antibody capable of capturing β-amyloid in the periphery.
(20) A monoclonal antibody that specifically reacts with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof and does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 8 against a mammal. A method of preventing or treating Alzheimer's disease, mild cognitive impairment or brain amyloid angiopathy, characterized by administering an effective amount;
(21) specifically reacts with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof for producing a prophylactic / therapeutic agent for Alzheimer's disease, mild cognitive impairment or brain amyloid angiopathy, and SEQ ID NO: 8 Use of a monoclonal antibody that does not recognize a partial peptide having the amino acid sequence represented,
(22) The antibody recognizes β-amyloid (1-42) having the amino acid sequence represented by SEQ ID NO: 5, but has β-amyloid (1-38) having the amino acid sequence represented by SEQ ID NO: 1. ), An antibody that does not recognize β-amyloid (1-39) having the amino acid sequence represented by SEQ ID NO: 2 and β-amyloid (1-40) having the amino acid sequence represented by SEQ ID NO: 3. (1) The agent described in the above is provided.

  By using a monoclonal antibody that specifically reacts with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof and does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 8, Β-amyloid having a region can be specifically recognized, and this antibody is useful as a prophylactic / therapeutic agent or diagnostic agent for Alzheimer's disease. Furthermore, since β-amyloid (x-42) has a low ratio in a soluble fraction such as cerebrospinal fluid, it is expected that the efficiency of export to the outside of the brain via the brain blood barrier is low. May selectively increase the blood concentration of only β-amyloid (x-42) and decrease insoluble brain β-amyloid (x-42). Moreover, since the said antibody hardly reacts with the cerebrovascular amyloid which has (beta) -amyloid (x-40) as a main component, it is thought that it does not cause the bleeding from the cerebral blood vessel containing a brain amyloid angiopathy.

  Among the hybridoma cells producing the anti-β-amyloid antibody used in the present invention, BA-27 has been established from December 22, 1992, as Fermentation Research Institute (IFO) (2-13 Jusohoncho, Yodogawa-ku, Osaka-shi, Osaka). 17-85 (postal code 532-8686) as IFO 50387, from 7 January 1993, National Institute of Advanced Industrial Science and Technology (NIBH), Ministry of International Trade and Industry It has been deposited as FERM BP-4139 at the Patent Biological Depositary Center (1-1-1 Chuo 6th, Tsukuba City, Ibaraki Prefecture, postal code 305-8666).

  Among the hybridoma cells producing the anti-β-amyloid antibody used in the present invention, BC-05 has been used since November 2, 1993, NIBH (National Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry) [currently, It is deposited as FERM BP-4457 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Higashi 1-1-1 Tsukuba, Ibaraki, Chuo 6th (zip code 305-8666)).

  In the present specification, the antibody obtained from each hybridoma cell is described in a form in which a is added to the cell name.

Among the SEQ ID NOs used in the present specification, SEQ ID NO: 1 to SEQ ID NO: 9 represent amino acid sequences of the following peptides.
[SEQ ID NO: 1] β-amyloid (1-38)
[SEQ ID NO: 2] β-amyloid (1-39)
[SEQ ID NO: 3] β-amyloid (1-40)
[SEQ ID NO: 4] β-amyloid (1-41)
[SEQ ID NO: 5] β-amyloid (1-42)
[SEQ ID NO: 6] β-amyloid (1-43)
[SEQ ID NO: 7] β-amyloid (1-28)
[SEQ ID NO: 8] β-amyloid (25-35)
[SEQ ID NO: 9] β-amyloid (35-43)

  In the present specification, the protein (polypeptide) has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end in accordance with the convention of peptide designation. The protein used in the present invention including the polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 may have any of a carboxyl group, a carboxylate, an amide, or an ester at the C-terminus.

  As β-amyloid in the present invention, β-amyloid (1-38) having the amino acid sequence represented by SEQ ID NO: 1, β-amyloid having the amino acid sequence represented by SEQ ID NO: 2 (1-39) Β-amyloid (1-40) having the amino acid sequence represented by SEQ ID NO: 3, β-amyloid (1-41) having the amino acid sequence represented by SEQ ID NO: 4, represented by SEQ ID NO: 5 Β-amyloid (1-42) having the amino acid sequence or β-amyloid (1-43) having the amino acid sequence represented by SEQ ID NO: 6 (preferably the amino acid sequence represented by SEQ ID NO: 5 Have β-amyloid (1-42) or the like.

  Examples of the β-amyloid derivative in the present invention include those in which the amino acid at the N-terminal part of β-amyloid is missing about 1 to 17 residues, L-aspartic acid is L-isoaspartic acid, D-isoaspartic acid or A peptide isomerized to D-aspartic acid, one having pyroglutamic acid at the N-terminal, and a peptide having an amino acid sequence lacking the first to tenth amino acids from the N-terminal of the β-amyloid, For example, glutamic acid converted to pyroglutamic acid is used. Specifically, a peptide having the second to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 5, and the third to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 5 A peptide having N-terminal glutamic acid converted to pyroglutamic acid, a peptide having the 4th to 42nd amino acid sequences of the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 1 to SEQ ID NO: 6 A peptide having an amino acid sequence lacking the first to 16th amino acid sequence or the first to 17th amino acid sequence from the amino acid sequence represented by (for example, β-amyloid (17-40), β-amyloid ( 18-40) etc. are used. These β-amyloids or derivatives thereof can be prepared from mammals such as humans, monkeys, rats, and mice by a method known per se, or commercially available natural purified preparations. A synthetic peptide may also be used.

  The partial peptide on the C-terminal side of β-amyloid or a derivative thereof in the present invention includes, for example, a partial peptide having an amino acid sequence from the 25th position counting from the N-terminal amino acid of β-amyloid.

  The aforementioned β-amyloid or a derivative thereof is, for example, a salt (preferably physiologically acceptable) with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal salt). Acid addition salts), and such salts include, for example, salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid). , Propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

As a monoclonal antibody that reacts specifically with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof in the present invention (hereinafter sometimes simply referred to as an antibody), for example, β-amyloid or a derivative thereof is recognized. An antibody that does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 7 (that is, a partial peptide on the N-terminal side of β-amyloid represented by β-amyloid (1-28)) is used. Further, a partial peptide that recognizes β-amyloid or a derivative thereof but has the amino acid sequence represented by SEQ ID NO: 8 (that is, a portion of the central portion of β-amyloid represented by β-amyloid (25-35)) An antibody that does not recognize peptide; a partial peptide that recognizes β-amyloid or a derivative thereof but has the amino acid sequence represented by SEQ ID NO: 9 (that is, β-amyloid represented by β-amyloid (35-43)) Or an antibody that does not recognize the partial peptide on the C-terminal side. More specifically, among these antibodies,
(I) an antibody that does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 8 and SEQ ID NO: 9 (that is, β-amyloid (25-35) and β-amyloid (35-43)),
(Ii) The partial peptide having the amino acid sequence represented by SEQ ID NO: 8 (that is, β-amyloid (25-35)) is not recognized, but the partial peptide having the amino acid sequence represented by SEQ ID NO: 9 (that is, An antibody that recognizes β-amyloid (35-43)) is preferred.

  Among the antibodies of (i) above, β-amyloid (1-38) having the amino acid sequence represented by SEQ ID NO: 1, β-amyloid having the amino acid sequence represented by SEQ ID NO: 2 (1-39) And / or an antibody that specifically recognizes β-amyloid (1-40) having the amino acid sequence represented by SEQ ID NO: 3 is preferred, and β-amyloid having the amino acid sequence represented by SEQ ID NO: 1 ( 1-38), β-amyloid (1-39) having the amino acid sequence represented by SEQ ID NO: 2 and β-amyloid (1-40) having the amino acid sequence represented by SEQ ID NO: 3 are recognized. An antibody that does not recognize β-amyloid (1-42) having the amino acid sequence represented by SEQ ID NO: 5 is preferable.

  Among the antibodies of (ii) above, β-amyloid (particularly β-amyloid (1-42) having the amino acid sequence represented by SEQ ID NO: 5) contained in the brain formic acid extract of Alzheimer's disease patients In which β-amyloid (1-42) having the amino acid sequence represented by SEQ ID NO: 5 is recognized but β-amyloid having the amino acid sequence represented by SEQ ID NO: 1 is preferred. (1-38), β-amyloid (1-39) having the amino acid sequence represented by SEQ ID NO: 2 and β-amyloid (1-40) having the amino acid sequence represented by SEQ ID NO: 3 are not recognized. Antibodies are preferred.

  As a typical example of the antibody of (i) above, there is a monoclonal antibody labeled with BA-27a (see WO 94/17197), and as a representative example of the antibody of (ii) above, BC-05a, BC There are monoclonal antibodies (see WO 94/17197) labeled with -15a, BC-65a, BC-75a, and BC-55a (particularly BC-05a is preferred).

As a method for preparing an antibody antigen and a method for producing an antibody, a method known per se, for example, the method described in WO 94/17197 or a method analogous thereto can be used. .
(1) Preparation of antigen Antigen used for preparing the antibody of the present invention includes, for example, β-amyloid or a derivative thereof, β-amyloid or a partial peptide obtained by hydrolyzing the derivative, β-amyloid, Any of synthetic peptides having one or more of the same antigenic determinants can be used (hereinafter, these may be simply referred to as β-amyloid antigen). As the β-amyloid or a derivative thereof, those described above are used. These β-amyloid or derivatives thereof can be prepared from mammals such as humans, monkeys, rats, and mice using a method known per se or a method analogous thereto, and are commercially available natural purified preparations. Also good. A synthetic peptide may also be used.

The β-amyloid or a derivative thereof or a salt thereof can be produced according to a known method, for example, a method described in WO 02/06483, and (a) a mammal such as a human, monkey, rat, mouse or the like. Prepared from animal tissues or cells using a known method or a method equivalent thereto, (b) chemically synthesized by a known peptide synthesis method using a peptide synthesizer, etc., (c) a polyamino acid containing a desired amino acid sequence It is also produced by culturing a transformant containing DNA encoding a peptide or a salt thereof.
(A) When preparing β-amyloid antigen from a tissue or cell of the mammal, the tissue or cell is homogenized and then extracted with an acid or alcohol, and the extract is subjected to salting out, dialysis, gel Purification and isolation can be performed by combining chromatography such as filtration, reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like.
(B) When β-amyloid antigen is chemically prepared, as the synthetic peptide, for example, one having the same structure as the above-described naturally purified β-amyloid antigen is used.
(C) When a polypeptide containing a desired amino acid sequence or a salt thereof is produced using a transformant containing DNA, the DNA can be obtained by a known cloning method [for example, Molecular Cloning (2nd ed .; J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). The cloning method includes (1) using a DNA probe or DNA primer designed based on the amino acid sequence of a polypeptide containing a desired amino acid sequence or a salt thereof, and containing the desired amino acid sequence from a cDNA library by a hybridization method. A method for obtaining a transformant containing a DNA encoding a polypeptide or a salt thereof, or (2) a PCR method using a DNA primer designed based on the amino acid sequence of a polypeptide or a salt thereof containing a desired amino acid sequence And a method for obtaining a transformant containing a DNA encoding a polypeptide containing a desired amino acid sequence or a salt thereof.

  As a partial peptide obtained by hydrolyzing the β-amyloid, for example, β-amyloid (1-43) having the amino acid sequence represented by SEQ ID NO: 6 is converted to N by exoprotease such as aminopeptidase or carboxypeptidase. Partial peptides obtained by sequential hydrolysis from the terminal and / or C-terminal or mixtures thereof, partial peptides obtained by hydrolyzing β-amyloid (1-43) with various endopeptidases, or mixtures thereof, etc. Is used. When β-amyloid (1-42) is produced by this method, β-amyloid (1-41) and / or β-amyloid (1-43) may be mixed in the preparation. Examples of the synthetic peptide include those having the same structure as the above-described naturally purified β-amyloid antigen and amino acid sequences such as β-amyloid (1-43) of 3 or more, preferably 6 or more amino acids. Peptides containing one or more amino acid sequences identical to the amino acid sequence at any location consisting of (hereinafter abbreviated as β-amyloid-related synthetic peptide) and the like are used.

  The synthetic peptide can be produced by known conventional means, and can be produced by either solid phase synthesis or liquid phase synthesis. That is, the peptide of interest can be produced by condensing a partial peptide or amino acid that can constitute the peptide and the remaining part, and removing the protecting group when the product has a protecting group. Specific examples of known condensation methods and protecting group elimination methods include, for example, B. Merrifield [J. Am. Chem. Soc., 85. 2149 (1963)], M. Bodanszky. And MA Ondetti (Peptide Synthesis, Interscience Publishers, New York, 1966), Schroder and Lubke (The Peptide, Academic Press, New York, 1965), Nobuo Izumiya et al. And Experiment, Maruzen, 1985], Haruaki Yajima and Shunpei Sugawara (Biochemistry Experiment Course 1, Protein Chemistry IV, 205, 1977). For example, when β-amyloid or a β-amyloid-related synthetic peptide is synthesized by a solid phase method, those known in the art as insoluble resins (for example, chloromethyl resin, 4-oxymethylphenylacetamidomethyl) Using any one of the resins, the protected amino acids are sequentially condensed from the C-terminal side of β-amyloid or β-amyloid-related synthetic peptide according to a conventional method. Next, all protecting groups are removed by treatment with hydrogen fluoride, and after purification by a method known per se such as high performance liquid chromatography, the target β-amyloid or β-amyloid-related synthetic peptide can be obtained. As the N-protected amino acid, the α-amino group is protected with a Boc group, and further, for example, the hydroxyl group of serine and threonine is protected with a Bzl group, and the ω-carboxyl group of glutamic acid and aspartic acid is protected with an OBzl group, The ε-amino group of lysine is protected with a Cl—Z group, the hydroxyl group of tyrosine is protected with a Br—Z group, the guanide group of arginine is protected with a Tos group, and the imidazole group of histidine is protected with a Bom group. Can be manufactured. Further, after the reaction, the peptide can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. .

  As the amide form of the peptide, a commercially available resin for peptide synthesis suitable for amide formation can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenyl. Examples include acetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. . Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target peptide. At the end of the reaction, the peptide is excised from the resin, and at the same time, various protecting groups are removed to obtain the desired peptide. Alternatively, a partially protected peptide can be taken out using chlorotrityl resin, oxime resin, 4-hydroxybenzoic acid resin, and the like, and the protective group can be removed by conventional means to obtain the target peptide.

  For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOBt, etc.), or a preprotected amino acid as a symmetric anhydride or HOBt ester or HOBt ester. Can be added to the resin after activation. The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents that are known to be usable for the peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and sulfoxides such as dimethyl sulfoxide. , Tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. The reaction temperature is appropriately selected from a range that is known to be usable for peptide bond formation reaction, and is usually selected from the range of about -20 ° C to about 50 ° C. The activated amino acid derivative is usually used in an excess of about 1.5 to about 4 times. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.

Examples of the protecting group for the amino group of the raw material amino acid include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoro Acetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like can be mentioned. Examples of the protecting group for the carboxyl group include a C _1-6 alkyl group, a C _3-8 cycloalkyl group, a C _7-14 aralkyl group, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, Examples include phenacyl and benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, and trityl hydrazide.

The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, groups derived from carbonic acid such as benzyloxycarbonyl groups and ethoxycarbonyl groups. . Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

  Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl-Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like.

  Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, Bom, Bum, Boc, Trt, Fmoc and the like.

  Examples of the activated carboxyl group of the raw material include the corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyano Methyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOBt)] and the like. Examples of the activated amino group of the raw material include the corresponding phosphoric acid amide.

  Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethane. Examples include acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia. The elimination reaction by the acid treatment is generally performed at a temperature of −20 ° C. to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, 1 It is effective to add a cation scavenger such as 1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide, dilute ammonia and the like.

  The protection of the functional group that should not be involved in the reaction of the raw material and the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.

  As another method for obtaining an amide form of a peptide, first, the α-carboxyl group of the carboxyl terminal amino acid is amidated, then the peptide chain is extended to the amino group side to the desired chain length, and then the N terminal of the peptide chain Of the peptide except for the protecting group of α-amino group and the peptide (or amino acid) except for the protecting group of the C-terminal carboxyl group, and condensing both peptides in a mixed solvent as described above. . The details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above method to obtain the desired crude peptide. This crude peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired peptide.

  To obtain an ester of a peptide, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired peptide can be obtained in the same manner as the peptide amide.

  Since β-amyloid antigen tends to aggregate, it can be directly immunized with insolubilized one. Alternatively, a complex in which the β-amyloid antigen is bound or adsorbed to an appropriate carrier may be immunized. The carrier and the mixing ratio of the carrier (carrier) and β-amyloid antigen (hapten) can be any ratio as long as the antibody can be efficiently produced against the β-amyloid antigen bound or adsorbed to the carrier. It may be bound or adsorbed with a natural or synthetic polymer carrier commonly used in the production of antibodies against hapten antigens and bound or adsorbed at a ratio of 0.1 to 100 with respect to hapten 1 Can be used. Natural polymer carriers include, for example, serum albumin from mammals such as cows, rabbits and humans, thyroglobulin from mammals such as cows and rabbits, hemoglobin from mammals such as cows, rabbits, humans and sheep, key Whole limpet hemocyanin is used. Examples of synthetic polymer carriers that can be used include various latexes such as polymers or copolymers of polyamino acids, polystyrenes, polyacryls, polyvinyls, polypropylenes, and the like.

Various coupling agents can be used for coupling the hapten and the carrier. For example, diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldehyde that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and thiol groups A dimaleimide compound such as N, N′-o-phenylenedimaleimide, a maleimide active ester compound that crosslinks an amino group and a thiol group, a carbodiimide compound that crosslinks an amino group and a carboxyl group, and the like are advantageously used. In addition, when cross-linking amino groups, an active ester reagent having a dithiopyridyl group on one of the amino groups (for example, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP)) was reacted. It is also possible to introduce a thiol group by post-reduction, introduce a maleimide group into the other amino group with a maleimide active ester reagent, and then react both.
(2) Preparation of monoclonal antibody β-amyloid antigen is administered to a warm-blooded animal by itself, for example, at a site where antibody production is possible or a carrier, by administration methods such as intraperitoneal injection, intravenous injection, and subcutaneous injection. Administered with diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chickens and the like, and mice, rats, etc. are preferably used for the production of monoclonal antibodies.

  When producing monoclonal antibodies, select warm-blooded animals immunized with β-amyloid antigen, for example, individuals with antibody titers from mice and collect spleen or lymph nodes 2-5 days after the final immunization. An anti-β-amyloid monoclonal antibody-producing hybridoma can be prepared by fusing the included antibody-producing cells with myeloma cells. The anti-β-amyloid antibody titer in serum is measured, for example, by reacting a labeled β-amyloid described below with antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but preferably PEG is used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc., and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to be used and the number of bone marrow cells is usually about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80%. In general, cell fusion can be carried out efficiently by incubating usually at 20 to 40 ° C., preferably 30 to 37 ° C. for usually 1 to 10 minutes.

  Various methods can be used for screening anti-β-amyloid antibody-producing hybridomas. For example, β-amyloid or β-amyloid-related synthetic peptide can be directly or adsorbed with a carrier on a solid phase (eg, microplate) on hybridoma culture. Add antiserum, then add anti-immunoglobulin antibody labeled with radioactive substances or enzymes (if the cell used for cell fusion is mouse, anti-mouse immunoglobulin antibody is used) or protein A and bind to the solid phase To detect the anti-β-amyloid monoclonal antibody, the hybridoma culture supernatant is added to the solid phase adsorbed with the anti-immunoglobulin antibody or protein A, β-amyloid labeled with a radioactive substance or an enzyme is added, and the solid phase is added. For detecting anti-β-amyloid monoclonal antibody bound to glycine Etc., and the like. Selection and breeding of anti-β-amyloid monoclonal antibody is usually carried out in an animal cell culture medium (eg, RPMI1640) containing 10-20% fetal calf serum with HAT (hypoxanthine, aminopterin, thymidine) added. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-described measurement of the anti-β-amyloid antibody titer in the antiserum.

  Separation and purification of anti-β-amyloid monoclonal antibody can be carried out in the same way as separation and purification of ordinary polyclonal antibodies (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchange) Absorption / desorption method by body (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A, protein G or KAPTIV-AE (TECNOGEN SCPA) A specific purification method for obtaining an antibody by dissociating the bond, etc.].

  Also, a hybridoma that produces an anti-β-amyloid antibody that reacts with a partial region of β-amyloid, and a hybridoma that produces an anti-β-amyloid monoclonal antibody that reacts with β-amyloid but does not react with the partial region This selection can be performed, for example, by measuring the binding property between the peptide corresponding to the partial region and the antibody produced by the hybridoma.

  As described above, the antibody of the present invention can be produced by culturing the hybridoma cell in vivo or ex vivo of a warm-blooded animal and collecting the antibody from the body fluid or culture.

  The thus obtained anti-β-amyloid monoclonal antibody can be used as a prophylactic / therapeutic agent (preferably an Alzheimer's disease) for Alzheimer's disease, mild cognitive impairment (MCI), brain amyloid angiopathy (CAA), etc. Can be used). Examples of the prophylactic / therapeutic agent include inhibitors of aggregation or deposition of β-amyloid in the brain, and agents that specifically increase the blood concentration of the peptide having the amino acid sequence represented by SEQ ID NO: 5. Preferably there is.

For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′ or Fab fraction of the antibody molecule may be used. The antibody of the present invention used for these purposes is preferably an antibody that permeates through the blood-brain barrier (BBB), and in particular, an antibody that suppresses the deposition of β-amyloid and an inhibitor of β-amyloid oligomerization. An antibody or an antibody that can extract β-amyloid from the formed senile plaques (or an antibody that can eliminate senile plaques formed in the brain) is preferred. Alternatively, it may be an antibody that does not permeate the brain blood barrier (BBB). In this case, an antibody that captures β-amyloid in the periphery at the periphery and promotes excretion of β-amyloid from the brain to the periphery. It is preferable that

In addition, antibodies that can penetrate the blood-brain barrier (BBB) and bind to β-amyloid in the formed senile plaque as described above are directly diagnosed in the body such as Alzheimer's disease and mild cognitive impairment (MCI). It can also be used as an agent. Diffuse senile plaques found in very early Alzheimer's disease and mild cognitive impairment (MCI) are known to have β-amyloid (x-42) as the main component, and therefore β-amyloid (x -42) antibodies specific to the C-terminal, F (ab ′) ₂ , Fab ′ or Fab fraction of the antibody molecule, and diffuse senile plaques directly in the body, such as high magnetic field MRI It is considered that the early diagnosis of Alzheimer's disease and mild cognitive impairment (MCI), which has been difficult until now, can be made possible by imaging using the image.

  The prophylactic / therapeutic agent for Alzheimer's disease, mild cognitive impairment (MCI), brain amyloid angiopathy (CAA) and the like (preferably Alzheimer's disease etc.) containing the antibody of the present invention has low toxicity, as it is as a liquid or as appropriate As a pharmaceutical composition of such a dosage form, it can be administered orally or parenterally to humans or mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). The dose varies depending on the administration subject, target disease, symptom, administration route and the like. For example, when used for the treatment of adult Alzheimer's disease, the antibody of the present invention is usually administered at a dose of 0.01 to About 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a week, preferably about 1 to 4 times a month, parenterally It is convenient to administer. In the case of oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

  The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the administration comprises the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration.

  For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

  As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. Is included. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-described antibody or a salt thereof with a normal suppository base.

The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually about 5 to 500 mg, especially about 5 to 5 mg for each dosage unit dosage form. It is preferable that 100 mg, other dosage forms contain about 10 to 250 mg of the antibody.
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.

In the specification of the present invention, when amino acids and the like are represented by abbreviations, they are based on abbreviations by IUPAC-IUB Commision on Biochemical Nomenclature or conventional abbreviations in the field. When there are optical isomers with respect to amino acids, L form is shown unless otherwise specified.
PAM: phenylacetamidomethyl Boc: t-butyloxycarbonyl Z: benzyloxycarbonyl Cl-Z: 2-chloro-benzyloxycarbonyl Bг-Z: 2-bromo-benzyloxycarbonyl Bzl: benzyl OcHex: cyclohexyl ester OBzl: Benzyl ester Tos: p-toluenesulfonyl HOBt: 1-benzotriazole MeBzl: 4-methylbenzyl Bom: benzyloxymethyl DCC: N, N′-dicyclohexylcarbodiimide TFA: trifluoroacetic acid DMF: N, N-dimethylformamide Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Guru Aspic acid Ass: Lys aspartate: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine SPDP: 3- (2-pyridyldithio) propionic acid N-Succinimidyl G MB N- (4-maleimidobutyryloxy) succinimide BSA: bovine serum albumin BTG: bovine thyroglobulin EIA: enzyme immunoassay HPLC: reverse phase high performance liquid chromatography HRP: horseradish peroxidase FBS: fetal bovine serum d-FBS: dialyzed Fetal bovine serum TMB: 3,3 ′, 5,5′-tetramethylbenzidine H / HBSS: Hepes buffered Hanks balance solution

The SEQ ID NOs used in the present specification represent the amino acid sequences of the following peptides.
[SEQ ID NO: 1] β-amyloid (1-38)
[SEQ ID NO: 2] β-amyloid (1-39)
[SEQ ID NO: 3] β-amyloid (1-40)
[SEQ ID NO: 4] β-amyloid (1-41)
[SEQ ID NO: 5] β-amyloid (1-42)
[SEQ ID NO: 6] β-amyloid (1-43)
[SEQ ID NO: 7] β-amyloid (1-28)
[SEQ ID NO: 8] β-amyloid (25-35)
[SEQ ID NO: 9] β-amyloid (35-43)

The present invention will be described in more detail below with reference to examples, but these do not limit the scope of the present invention.
The monoclonal antibodies BC-05a and BA-27a used in Examples were obtained according to the method described in Example 7 of WO 94/17197.

(1) Measurement of Aβx-40 and Aβx-42 by sandwich method-EIA Mouse monoclonal antibody BNT-77a (Asami-Odaka A. et al., Biochemistry, 34, 10272-10278, 1995) is WO 94 According to the method described in Example 7 of Japanese Patent No. 17717, β-amyloid (11-28) was obtained by immunizing BALB / C mice. 100 μl each of 0.1 M carbonate buffer solution and pH 9.6 solution containing 15 μg / ml was dispensed into a 96-well microplate and left at 4 ° C. for 24 hours. Excess binding sites in the wells were inactivated by adding 300 μl of Block Ace (Dainippon Pharmaceutical Co., Ltd.) diluted 4-fold with PBS and leaving at 4 ° C. for 24 hours. For the measurement of Aβx-40, a buffer EC [10% Block Ace, 0.2% BSA, 0.4M NaCl, 0.05% CHAPS, 2 mM EDTA, 0.05% NaN ₃ is contained on the plate on which the primary antibody is immobilized as described above. A dilution series of Aβ1-40 (Peptide Laboratories) diluted with 0.02M phosphate buffer, pH 7] and 100 μl of the test sample were added and reacted at 4 ° C. for 24 hours. After washing with PBS, BA-27a-HRP (WO 94 /) diluted as a secondary antibody with buffer C [0.02M phosphate buffer, pH 7 containing 1% BSA, 0.4M NaCl, and 2 mM EDTA] 1000 times. 17197) was added and reacted at room temperature for 6 hours. After washing with PBS, 100 μl of TMB microwell peroxidase substrate system (KIRKEGAARD & PERRY LAB, INC) was added and allowed to react at room temperature for 10 minutes. After the reaction was stopped by adding 100 μl of 1M phosphoric acid, the absorbance at 450 nm was measured with a plate reader (SPECTRAMAX190, Molecular Device) to determine the enzyme activity on the solid phase. Aβx-42 was measured by adding a dilution series of Aβ1-42 (Peptide Institute) and a test sample to the BNT-77a-immobilized plate prepared above, and using BC-05a-HRP (WO 94/17197 as a secondary antibody). The measurement was performed in the same manner as Aβx-40.

(2) Changes in Aβx-40 and Aβx-42 concentrations in BALB / C mouse plasma and cerebrospinal fluid after intraperitoneal administration of BA-27a or BC-05a
The effect of the antibody was examined in 19 BALB / C mice at 3 months of age. 0.5 mg BA-27a and 0.5 mg BC-05a were administered intraperitoneally to 7 and 6 mice, respectively. The remaining 6 untreated animals were examined as controls. Sixteen hours after administration, plasma and cerebrospinal fluid (CSF) were collected from mice under ether anesthesia, and Aβx-40 and Aβx-42 were measured. Furthermore, plasma of mice administered with BC-05a intraperitoneally was collected 4 times every other week, and Aβx-42 was measured. Of these, 10 μl of plasma and 5 μg of Dynabeads M280 anti-mouse Ig (Dynal) were reacted at room temperature for 2 hours, and then the supernatant was collected, and non-immunoglobulin-bound Aβx-40, Aβx-42 and 0.2 M in plasma were collected. Aβx-40 and Aβx-42 were measured for the immunoglobulin-bound forms eluted with Glycine pH2.8.
Plasma Aβx-40 was 137.3 ± 2.2 fmol / ml in the BA-27a administration group, 3.3 ± 1.2 fmol / ml in the BC-O5a administration group, and 3.4 ± 1.8 fmol / ml in the normal control group.
Cerebrospinal fluid Aβx-40 was 670.8 ± 252.6 fmol / ml in the BA-27a administration group, 166.5 ± 140.1 fmol / ml in the BC-O5a administration group, and 246 ± 184.3 fmol / ml in the normal control group. Plasma Aβx-42 was 0.5 ± 0.4 fmol / ml in the BA-27a administration group, 60.7 ± 7.1 fmol / ml in the BC-O5a administration group, and 0.2 ± 0.2 fmol / ml in the normal control group.
Cerebrospinal fluid Aβx-42 was below the measurement sensitivity in the BA-27a administration group, 0.6 ± 0.4 fmol / ml in the BC-O5a administration group, and below the measurement sensitivity in the normal control group.
Aβx-40 was significantly increased by 40-fold in plasma and 2.7-fold in cerebrospinal fluid by administration of BA-27a (p <0.001). BC-05a administration did not increase Aβx-40 in both plasma and cerebrospinal fluid.
Aβx-42 was not elevated in both plasma and cerebrospinal fluid when BA-27a was administered. BC-05a administration significantly increased the plasma by a factor of 304 (p <0.001), while cerebrospinal fluid had a sensitivity lower than that of the normal control but increased to 0.6 ± 0.4 fmol / ml.
In mice that continued to receive BC-05a for one month, plasma Aβx-42 increased to 324.1 fmol / ml, a 1621-fold increase. In addition, antibody-bound and antibody-unbound were separated by immunoprecipitation. Then, almost 100% of the increased Aβx-42 was non-antibody-bound.
The above results indicate that cerebrospinal fluid Aβx-42 and plasma Aβx-42 are specifically and markedly elevated when anti-Aβ42C end-specific antibody BC-05a is administered to the peritoneal cavity of mice. Since Aβx-42 elevated in plasma is non-immunoglobulin-bound, this increase in Aβx-42 is not a measure of Aβx-42 to which the administered BC-05a is bound in blood.
Therefore, it is considered that administered BC-05a specifically increased the clearance of Aβx-42 in the brain, resulting in an increase in cerebrospinal fluid Aβx-42 and plasma Aβx-42.
This result shows that administration of BC-05a can selectively control the concentration of Aβx-42 in the brain, which is the onset factor of Alzheimer's disease, and brain amyloid Aβx, which is deposited once and causes various pathological processes. This shows that -42 deposition can be selectively released and improved. Applying the combination of administered BC-05a with Aβx-42 in the brain, selective imaging of senile plaques, selective estimation of brain amyloid Aβx-42 deposition by intravascular administration of BC-05a, etc. It can be applied to new diagnostic methods.

(1) Preparation of biotinylated BC-05a and biotinylated mouse IgG
To 2 ml of BC-05a (10 mg / ml), 40 μl of an aqueous solution of sulfo-NHS-biotin (Pierce) (2.4 mg / 120 μl) was added and reacted at room temperature for 30 minutes with stirring. The reaction solution was diluted and dialyzed against PBS (−) at 4 ° C. for 2 days, and then the antibody concentration was measured. Yield about 70%. Mouse IgG (Wako Pure Chemical Industries) used as a control was biotinylated in the same manner.
(2) Short-term continuous injection of biotinylated antibody to young APPswTg (Tg2576) mice, preparation of soluble fraction of cerebrum and measurement of intracerebral antibody concentration Female Tg2576 mice (12 weeks old) (Science, 274, 99-102) 1996), biotinylated mouse IgG or biotinylated BC-05a (0.5 mg / 0.2 ml / mouse each) was administered once intraperitoneally, blood was collected 24 hours later, cerebral was collected after perfusion, and frozen after half-cutting (n = 4-5). To the blood, protease inhibitor cocktail (Complete, Roche) and EDTA (final concentration 4 mg / ml) were added and mixed, and the supernatant after centrifugation was stored frozen as plasma. After adding 50 mM Tris-HCl containing physiological saline (pH 7.5) (TS buffer) containing protease inhibitor cocktail to the cerebral hemisphere and adding 4 times its weight to homogenization, ultracentrifugation at 300,000 xg for 20 minutes at 4 ° C Kiyoshi was used as a soluble fraction of cerebrum. This was added to an avidin-immobilized 96-well plate (Reacti-Bind NeutrAvidin Coated Plate, Pierce) and reacted at 4 ° C. overnight. After washing, anti-mouse IgG-HRP (Amersham) was added and reacted at room temperature for 6 hours. After washing, an HRP substrate (TMB Microwell Peroxidase substrate system, KPL) was added. After stopping the reaction with 1M phosphoric acid, the absorbance at 450 nm was measured with a plate reader, and the antibody concentration in the brain was calculated with the attached calculation software (SoftMAX). The antibody concentration in plasma was also measured in the same manner. A calibration curve obtained by using BC-05a as a standard was prepared, and the plasma and brain concentration results of biotinylated BC-05a obtained in (1) above are shown in Table 1.
[Table 1]

Biotinylated mouse IgG administration group Biotinylated BC-05a administration group
(N = 4) (pmol / g wet tissue) (n = 5) (pmol / g wet tissue)
Plasma 1300 ± 296 1030 ± 94.8
Cerebral soluble fraction 6.42 ± 0.475 4.75 ± 0.056
mean ± SEM

This shows that about 0.5% of the antibody concentration in plasma is transferred into the brain when either biotinylated BC-05a or biotinylated IgG is administered.

BC-05a to young APPswTg (Tg2576) mice for 9 months, preparation of cerebral extract and quantification of blood and brain Aβ concentration Male Tg2576 mice (10 weeks old) (Science, 274, 99-102) , 1996) mouse IgG or BC-05a (0.5 mg / 0.2 ml / mouse each until 15 weeks of age, 1.0 mg / 0.2 ml / mouse after 16 weeks of age) once a week, 52 weeks of age (12 Intraperitoneally (until 9 months) (n = 9-10). Sampling was performed in the same manner as described above.
Cerebral extract was prepared by separating the soluble fraction and the insoluble fraction. The soluble fraction was prepared in the same manner as in Example 2, and the precipitate after ultracentrifugation was washed with TS buffer, and then 8 times the cerebral hemisphere weight of 6 mM guanidine hydrochloride-containing 50 mM Tris-HCl solution (pH 7.5) was added. Added and dissolved. Centrifugation was performed at 15000 rpm and 4 ° C. for 20 minutes, and the supernatant was used as an insoluble fraction. The Aβ concentration was measured according to the method described in Example 1. The cerebral insoluble fraction was diluted 2000 times with buffer EC and used as a sample.
The Aβ concentration in plasma was determined in two ways: a method of directly quantifying diluted plasma, and a method of quantifying the diluted product after dissociating the BC-05a complex with guanidine hydrochloride. The latter was prepared by adding 30 μl of 50 mM Tris-HCl aqueous solution (pH 7.5) containing 8 M guanidine hydrochloride to 10 μl of plasma, mixing, and then diluting by adding 560 μl of buffer EC to make an ELISA sample.
The results are shown in Table 2.
[Table 2]

Aβx-40 Aβx-42
(Pmol / g wet tissue) (pmol / g wet tissue)
Plasma (no treatment) IgG administration group 3160 ± 302 389 ± 20.0
BC-05a administration group 2270 ± 186 8810 ± 224
Plasma (guanidine treated) IgG administration group 2990 ± 208 588 ± 29.0
BC-05a administration group 2350 ± 128 18800 ± 1100
Cerebral soluble Aβ IgG group 2.23 ± 0.402 0.413 ± 0.0521
BC-05a administration group 2.29 ± 0.27 0.643 ± 0.0687
Cerebral insoluble Aβ IgG group 1650 ± 383 645 ± 102
BC-05a administration group 1200 ± 347 442 ± 57.4
mean ± SEM

Similar to the single administration, Aβ concentration in plasma was greatly increased from 22 to 32 times by BC-05a administration. Treatment of plasma with guanidine increased the plasma Aβx-42 concentration in the BC-05a administration group by a factor of about 2; therefore, BC-05a may contribute to the stabilization of Aβx-42 in the blood. Sex is conceivable.
There was no change in the soluble Aβx-40 concentration in the brain, but the insoluble Aβx-40 concentration showed a decreasing trend. The concentration of Aβx-42 in the brain increased significantly in the soluble fraction and decreased in the insoluble fraction.

Claims (22)

Alzheimer's disease, mild cognition comprising a monoclonal antibody that specifically reacts with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof and does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 8 Preventive or therapeutic agent for disorders or brain amyloid angiopathy. The agent according to claim 1, which is a prophylactic / therapeutic agent for Alzheimer's disease. The agent according to claim 1, wherein the antibody does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 9. The agent according to claim 1, wherein the antibody is an antibody that recognizes a partial peptide having the amino acid sequence represented by SEQ ID NO: 9. The agent according to claim 1, wherein β-amyloid is a peptide having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. . The agent according to claim 1, wherein β-amyloid is a peptide having an amino acid sequence represented by SEQ ID NO: 5. A derivative of β-amyloid is a peptide having the 2nd to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 5, and the 3rd to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 5. The agent according to claim 1, which is a peptide having N-terminal glutamic acid converted to pyroglutamic acid or a peptide having the fourth to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 5. The β-amyloid derivative is a peptide having an amino acid sequence lacking the first to tenth amino acid sequences from each of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 6, and the N-terminal glutamic acid is pyro The agent according to claim 1, which is a peptide converted to glutamic acid. The agent according to claim 1, wherein the partial peptide on the C-terminal side of β-amyloid or a derivative thereof is a partial peptide having an amino acid sequence of the 25th and subsequent positions counted from the N-terminal amino acid of each β-amyloid. The agent according to claim 1, wherein the antibody does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 7. The agent according to claim 1, wherein the antibody is an antibody recognizing β-amyloid (1-42) having the amino acid sequence represented by SEQ ID NO: 5. The agent according to claim 1, wherein the antibody is a monoclonal antibody BA-27a that can be produced from a hybridoma cell labeled with BA-27 (FERM BP-4139). The agent according to claim 1, wherein the antibody is a monoclonal antibody BC-05a that can be produced from a hybridoma cell labeled with BC-05 (FERM BP-4457). The agent according to claim 1, wherein the antibody is an antibody that permeates the brain blood barrier. The agent according to claim 14, wherein the antibody is an antibody capable of extracting β-amyloid from the formed senile plaques. The agent according to claim 1, which is an inhibitor of aggregation or deposition of β-amyloid in the brain. The agent according to claim 1, which can specifically increase the blood concentration of the peptide having the amino acid sequence represented by SEQ ID NO: 5. The agent according to claim 1, wherein the antibody is an antibody that does not penetrate the brain blood barrier. The agent according to claim 18, wherein the antibody is an antibody capable of capturing β-amyloid in the periphery. An effective amount of a monoclonal antibody that specifically reacts with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof and does not recognize a partial peptide having the amino acid sequence represented by SEQ ID NO: 8 for mammals. A method for the prophylaxis or treatment of Alzheimer's disease, mild cognitive impairment or brain amyloid angiopathy characterized by administration. It specifically reacts with a partial peptide on the C-terminal side of β-amyloid or a derivative thereof for producing a prophylactic / therapeutic agent for Alzheimer's disease, mild cognitive impairment or brain amyloid angiopathy, and is represented by SEQ ID NO: 8 Use of a monoclonal antibody that does not recognize a partial peptide having an amino acid sequence. The antibody recognizes β-amyloid (1-42) having the amino acid sequence represented by SEQ ID NO: 5, but β-amyloid (1-38) having the amino acid sequence represented by SEQ ID NO: 1, sequence The antibody which does not recognize β-amyloid (1-39) having the amino acid sequence represented by No: 2 and β-amyloid (1-40) having the amino acid sequence represented by SEQ ID No: 3. Agent.