JP2007077103A - Prophylactic or therapeutic agent for alzheimer's disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a prophylactic or therapeutic agent for Alzheimer's disease having reduced adverse effects but nevertheless exhibiting high prophylactic or therapeutic effects. <P>SOLUTION: The prophylactic or therapeutic agent for the Alzheimer's disease comprises the following as an active ingredient. an F(ab')<SB>2</SB>fragment of an anti-β-amyloid peptide antibody or a fragment which is a part thereof and contains at least a variable region thereof or a conjugate of the fragment maintaining a binding ability to the β-amyloid peptide. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a preventive or therapeutic agent for Alzheimer's disease.

  Formation of β-amyloid peptide (hereinafter sometimes referred to as “Aβ”) plaques in the brain plays a central role in neurodegeneration that is characteristic of Alzheimer's disease (Non-patent Documents 1 and 2). Amyloid deposition can be reduced in a mouse Alzheimer's disease model by active administration of Aβ peptide and systemic administration of anti-Aβ monoclonal antibody (hereinafter, monoclonal antibody may be referred to as “mAb”) (non-patented) Documents 3 to 6) have been reported to improve cognitive behavior (Non-patent Documents 7 and 8). Furthermore, it has been reported more recently that intra-ventricular administration of an anti-Aβ antibody reduces amyloid deposition in the brain and the associated neuropathological symptoms in Tg mice, a model of Alzheimer's disease ( Non-patent document 9).

Yankner BA, Duffy LK, Kirschner DA. 1990. Neurotrophic and neurotoxic effects of amyloid beta protein: Reversal by tachykinin neuropeptides. Science 250: 279-282. Hyman BT. 1998. New neuropathological criteria for Alzheimer disease. Arch. Neural. 55: 1174-1176. Schenk D, Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko., D., Lee, M., Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C. , Walker, S., Wogulis, M., Yendnock, T., Games, D., Seubert, P. 1999. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.Nature 400. Bard F, Cannon, C., Barbour, R., Burke, RL, Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan , K., Kholodenko, D., Lee, M., Lieberberg, I., Motter, R., Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B. 2000. Peripherally administered antibodies against amyloid b -peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer's disease. Nat. Med. 6: 916-919. DeMattos RB, Bales, KR, Cummins, DJ, Dodart, JC, Paul, SM, Holtzman, DM 2001.Peripheral anti-Ab antibody alters CNS and plasma Ab clearance and decreases brain Ab burden in a mouse model of Alzheimer's disease.Proc. Natl. Acad. Sci. USA. 98: 8850-8855. Bard F, Barbour, R., Cannon, C., Carretto, R., Fox, M., Games, D., Guido, T., Hoenow, K., Hu, K., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, C., Lee, M., Motter, R., Nguyen, M., Reed, A., Schenk, D., Tang, P., Vasquez, N., Seubert, P., Yednock, T. 2003. Epitope and isotype specificities of antibodies to beta-amyloid peptide for protection against Alzheimer's disease-like neuropathology. Proc Natl. Acad. Sci. USA. 100: 2023-2028. Dodart JC, Bales, KR, Gannon, KS, Greene, SJ, DeMattos, RB, Mathis, C., DeLong, CA, Wu, S., Wu, X., Holtzman, DM, Paul, SM 2002. Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat. Neurosci. 5: 452-457. Schenk D, Yednock, T. 2002.The role of microglia in Alzheimer's disease: Neurobiol Aging 23: 677-679. Chauhan NB, Siegel GJ. 2003. Intracerebroventricular passive immunization with anti-Ab antibody in Tg2576. J. Neurosci. Res 74: 142-147. Check E. 2002. Nerve inflammation halts trial for Alzheimer's drug.Nature 415: 462. Hock C, Konietzko, U., Parassotiropoulos, A., Wollmer, A., Streffer, J., Von Rotz, RC, Davey, G., Moritz, E., Nitsch, RM 2002. Generation of antibodies specific for bete- amyloid by vaccination of patients with Alzheimer disease. Nat. Med. 8: 1270-1275.

  However, the prevention or treatment of Alzheimer's disease by active immunity or passive immunity described above has side effects. Although active immunization was conducted until clinical trials, meningoencephalitis occurred in some patients and the clinical trials were discontinued (Non-patent Documents 10, 11, and 8).

  Therefore, an object of the present invention is to provide a preventive or therapeutic agent for Alzheimer's disease, which has reduced side effects and can still exhibit a high preventive or therapeutic effect.

As a result of diligent research, the inventors of the present application have found that the F (ab ′) ₂ fragment of the anti-Aβ monoclonal antibody exhibits the same excellent preventive or therapeutic effect as the anti-Aβ monoclonal antibody, but has side effects of inflammation. As a result, the present invention was completed.

That is, the present invention provides an F (ab ′) ₂ fragment of an anti-β-amyloid peptide antibody, a fragment that is a part of the fragment and includes at least the variable region, or a conjugate of the fragment, and a β-amyloid peptide The present invention provides a preventive or therapeutic agent for Alzheimer's disease containing an active ingredient that maintains the binding ability of

  According to the present invention, a novel prophylactic or therapeutic agent for Alzheimer's disease that can exhibit a high preventive or therapeutic effect and yet has reduced side effects has been provided.

As described above, the preventive or therapeutic agent for Alzheimer's disease of the present invention is an F (ab ′) ₂ fragment of an anti-β-amyloid peptide antibody, or a fragment that is a part of the fragment and includes at least the variable region thereof, A binding substance that maintains the ability to bind to β-amyloid peptide is contained as an active ingredient. Here, examples of the fragment that is a part of the F (ab ′) ₂ fragment and includes at least the variable region thereof include Fab fragments and Fv fragments. In addition, these conjugates include scFv fragments, bivalent to tetravalent antibodies in which 2 to 4 Fv fragments are bound, Fab dimers and trimers in which 2 or 3 Fab fragments are bound, etc. Can be illustrated. These fragments and conjugates thereof are well known in the field of antibody medicine. In addition, since antibody fragments can be produced by genetic engineering techniques, they can be produced by genetic engineering techniques as long as they contain the variable region in the F (ab ′) ₂ fragment and have the ability to bind Aβ. It is also possible to use any fragment or its conjugate. Of these, the F (ab ′) ₂ fragment is most preferable from the viewpoint of drug efficacy.

The F (ab ′) ₂ fragment and the like used in the present invention may be derived from a polyclonal antibody, but a fragment derived from a monoclonal antibody is preferable from the viewpoint of enhancing the reproducibility of the drug effect. Anti-Aβ mAb immunizes an animal with Aβ or an antigenic fragment thereof, produces a hybridoma that produces a monoclonal antibody by a conventional method, and produces a monoclonal antibody from the culture supernatant or ascites of the animal administered the hybridoma intraperitoneally It can obtain by collect | recovering. Since the base sequence and amino acid sequence of Aβ are known, Aβ or a fragment thereof having antigenicity can be easily prepared by a peptide synthesizer or a genetic engineering technique. The amino acid sequence of human Aβ is shown in SEQ ID NO: 1 in the sequence listing. In Examples described later, a polypeptide (SEQ ID NO: 2) consisting of amino acid residues 1 to 42 from the N-terminus of human Aβ (SEQ ID NO: 1) is chemically synthesized by a peptide synthesizer, and this is used as an immunogen to immunize animals. did. When an Aβ fragment is used as an immunogen, the number of amino acid residues is preferably 20 or more, more preferably 30 or more, in order to ensure sufficient antigenicity and specificity. After obtaining a monoclonal antibody, an antibody fragment used as an active ingredient can be obtained by a conventional method such as enzyme treatment. For example, F (ab ′) ₂ fragment can be obtained by treating a monoclonal antibody with pepsin according to a conventional method.

  The preventive or therapeutic agent for Alzheimer's disease of the present invention is effective for both prevention and treatment of Alzheimer's disease. In the present invention, “treatment” includes preventing the progression of mild Alzheimer's disease more severely or delaying its progression rate.

  The route of administration of the preventive or therapeutic agent for Alzheimer's disease of the present invention is not particularly limited, but parenteral administration is preferable, and systemic administration such as intravenous injection and local administration to the brain such as intraventricular administration are particularly preferred. Is preferred. The dose is appropriately set according to the patient's symptoms and purpose of administration (prophylaxis or treatment, etc.), the type of active ingredient, etc., but usually 2.5 mg to 2500 mg as the amount of active ingredient per patient per day, Preferably, it is about 25 mg to 250 mg.

  The antibody fragment of the active ingredient can be prepared by a method commonly used in the pharmaceutical preparation field to prepare the preventive or therapeutic agent for Alzheimer's disease of the present invention. For example, the antibody fragment of the active ingredient can be dissolved in an injectable carrier (for example, a phosphate buffer solution) and formulated into an injectable solution. In the case of an injection solution, the concentration of the antibody fragment in the injection solution is not particularly limited, but usually about 250 mg / L to 2500 mg / L is appropriate.

  As specifically shown in the following examples, the preventive or therapeutic agent for Alzheimer's disease of the present invention exhibits a preventive or therapeutic effect comparable to a known method using all antibodies, and still serves as an index of inflammation. There are very few CD11b-positive cells occurring in brain tissue. Therefore, the prophylactic or therapeutic agent for Alzheimer's disease of the present invention can exhibit the same high prophylactic or therapeutic effect as known prophylactic or therapeutic agents, and yet has reduced side effects.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Materials and methods
Preparation of Aβ-specific hybridoma A peptide containing human Aβ 1-42 amino acids (Aβ _1-42 ) was synthesized and immunized 3 times at 100 μg per BALB / c mouse. That is, for the first immunization, Freund's complete adjuvant was mixed with the synthesized peptide, and after 2 weeks from the initial immunization, Freund's incomplete adjuvant was mixed with the peptide and boosted. After 3 weeks from the booster, after the final immunization with peptide alone, the antibody titer in serum against A [beta] _1-13 was excised spleens of high titers of mice. Lymphocytes were collected from the excised spleen and cell fusion was performed by a conventional method to produce anti-Aβ _1-13 antibody-producing hybridoma cells.

Culture of anti-Aβ _1-42 antibody-producing hybridoma cells Anti-Aβ _1-13 antibody-producing hybridoma cells (hereinafter abbreviated as IIA2) in KBM 450 (Kohjin Bio Co., Ltd, Saitama, Japan) in serum-free medium Culture was performed. After the culture, the culture supernatant was collected and subjected to salting out precipitation with ammonium sulfate to roughly purify the anti-Aβ _1-13 antibody in the culture supernatant. Antibodies crude is Immunopure F (ab ') obtained by pepsin treatment ₍₂ fragments and pFc' fragments (whole antibody _{2 preparation kit (Pierce Biotechnology, Rockford} IL, USA) F ab) using ', F ( ab ′) The other fragment different from the ₂ fragment was purified. That is, 1 ml of the sample adjusted to 5 mg / ml was cleaved with pepsin at 37 ° C. for 6 hours. After cleavage, F (ab ′) ₂ and pFc ′ and IgG that had not been cleaved were separated from the reaction solution using a Protein A column. Of these, the F (ab ′) ₂ fragment was recovered from the column and was used against phosphate buffer (PBS) using a tube with a cutoff molecular weight of 50,000 (MWCO: 50,000, Spectrum Medical Industries, Inc. CA, USA). Dialyzed. IgG and pFc ′ fragments that were not cleaved with pepsin were separated on a Sephadex G-200 column (trade name). These purified F (ab ′) ₂ and pFc ′ were used for in vivo experiments.

ELISA
Measurement of anti-Aβ _1-13 antibody in the culture supernatant of hybridoma cells was performed using ELISA. ELISA was performed by Okuda et al. (Okuda K, Kaneko T. 1993.Strong synergistic effects of polyvalent vaccine using synthetic peptide for human immunodeficiency virus.AIDS Res Hum Retrov 9 Suppl: S114; Okuda K, Bukawa H, Kawamoto S, Imai M, Saito T, Phanuphak P, Hamajima K. 1994.A serologic analysis and the amino acid sequence of the V3 region of human immunodeficiency virus from carriers in Bangkok.J Infect Dis 169: 227-228.) HIVIIIB env peptide (IRIQRGPGRAFVTIGKIGN) and myoglobin peptide (ISEAIIHVLHSRHP) were immobilized as a negative control and 40 μg / ml Aβ _1-42 peptide dissolved in 0.15 M PBS in a microtiter plate. The solid-phased plate was washed with PBS and blocked with 3% bovine serum albumin (BSA) for 1 h. After the blocking, the hybridoma cell culture supernatant was reacted at 4 ° C. for 6 h. After completion of the reaction, detection was carried out by reacting with horseradish peroxidase (HRP) -labeled anti-mouse IgG (Pierce Chemicals).

MAb adsorption experiments were carried out by incubating beads coated with Aβ _1-42 or its partial peptide used for animal immunization and anti-Aβ mAb. The peptide-coated beads were reacted with mAb at 4 ° C. for 12 hours. The HIV peptide or myoglobin peptide was used as a control. The antibody titer was then measured by ELISA.

In vivo treatment experiments using transgenic mice
Tg 2576 mice expressing human Aβ (Hsiao K, Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y., Younkin, S., Yang, F., Cole, G. 1996. Correlative Memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274: 99-102.) were obtained from Taconic Farms Inc. (German Town, NY, USA). Adjusted to 0.5 mg / ml in 65-week-old mice (solvent is Dulbeccos PBS (-), the same applies below) 0.5 ml of p-AβmAb (hereinafter “p-” means purification, “pp” means partial purification) PF (ab ′) ₂ and p-pFc ′ (6 to 7 mice in each group) were administered intraperitoneally. The brains were removed when these mice were 80 weeks old. In addition, the same treatment was applied to mice aged 60-70 weeks and 75-85 weeks. The left side of the excised brain was used for immunostaining, and the right side was used for measuring the amount of Aβ in the brain by ELISA.

Furthermore, treatment was performed by directly injecting 0.02 ml of p-AβmAb, pF (ab ′) ₂ and p-pFc ′ adjusted to 0.5 mg / ml into the frontal cortex on the left and right in the brain. After the treatment, the left hemisphere of the mouse that had passed for 5 days was cut with a cryostat microtome, and the right hemisphere was used for measuring the amount of Aβ.

Preparation of mouse brain specimen The left hemisphere of the removed brain was embedded in Tissue-Tek OCT Compound (trade name, Sakura Finetechnical Co., Ltd., Tokyo, Japan), and the cerebellum was removed from the right hemisphere. These brains were rapidly frozen in dry ice-acetone and stored at −80 ° C. until use.

Mesenamine silver staining Mesenamine silver staining was used to detect Aβ in the hippocampus and cerebral cortex in the brain. Staining was performed according to the method of Haga et al.

Fluorescent staining of Aβ in the brain The left brain of a mouse treated with a monoclonal antibody was removed, a frozen section was prepared with a cryostat, and fluorescent staining of Aβ was performed. For fluorescent staining, an avidin / biotin blocking kit (NICHIREI CORPORATION. Tokyo, JAPAN) was used to block endogenous non-specific reactions. Thereafter, the anti-human A [beta] ₁ - ₄₂ Rabbit antibody (IBL Co., Ltd. Gunma, Japan ), biotin-labeled anti-rabbit IgG antibody (MBL Co., Ltd. Nagoya, Japan ) and streptavidin fluorescent dye (FITC) (MBL Co ., Ltd.). At the same time, FITC-labeled A [beta] ₁ was self adjusting - direct staining with ₄₂ rabbit IgG antibody was also performed. After fluorescent staining, observation was performed with an OLYMPUS microscope BX60 connected to a confocal laser scanning microscope (Carl Zeiss LSM510) equipped with a high-sensitivity CCD camera. Moreover, A [beta] ₁ - ₄₂ simultaneously with microglial cells (Microglial cells) staining anti-mouse CD11b (Mac-1), PE (phycoerythrin, Ebioscience Inc. CA, USA) labeled monochrome - monoclonal antibody and PE- anti-mouse IA ^b MHC Class II antibody (ebioscience Inc. CA, USA) was used.

Measurement of brain Aβ protein by ELISA To measure the level of Aβ protein in the brain, ELISA was performed. ELISA is the method of Kawarabayashi et al. (Kawarabayashi T, Younkin, LH, Saido, CT, Shoji, M., Hsiao, K., Younkin, S. 2001.Age-dependent changes in brain, CSF, and Plasma Amyloid b protein in the Tg2576 transgenic mouse model of Alzheimer's disease. J. Neurosci. Res 21: 372-381.). That is, the frozen right brain was crushed with an ultrasonic wave and centrifuged. After centrifugation, the sediment was crushed again with ultrasonic waves, and a Tris buffer containing 1% triton x-100 (trade name) was added. The extracted Aβ was dissolved in 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA). After lysis, Aβ protein was quantified using a human Amyloid β assay kit (IBL Co., Ltd. Gunma, JAPAN).

Aβ F (ab ') ₂ intraperitoneal A [beta] plug in the brain after administration - click anti Aβ F (ab') ₂ Detection of fragment coupling
We investigated whether Aβ F (ab ′) ₂ administered intraperitoneally could enter the brain. For the examination, F (ab ′) ₂ fragment prepared from Aβ monoclonal antibody (IIA2) was subjected to biotin labeling (Biotinyl-N-hydroxysuccimide, COSMO BIO CO., LTD Tokyo. Japan). That is, 1 ml of the purified fragment adjusted to 1 mg / ml was dissolved in 0.1 M sodium carbonate and mixed with 60 μl of 1 mg / ml DMSO. The mixed purified fragment and DMSO were stirred at room temperature for 4 h. After stirring, the mixture was adjusted with PBS to a final concentration of 1 mg / ml, and 0.5 ml was administered intraperitoneally to 60-week-old Tg2576 mice. After administration, the mouse brain after 4 hours was removed and a frozen section was prepared. This section was treated with streptavidin FITC, and Aβ in the brain was detected under a fluorescence microscope.

result
Distribution of Aβ in the brain of Tg2576 mice FIG. 1 shows the distribution of Aβ plaques in the brain of transgenic Tg2576 mice expressing human Alzheimer's disease. In 80-week-old mice, many Aβ plaques were found in the hippocampus and cerebral cortex in the brain.

Analysis of Anti-Aβ Monoclonal Antibody IIA2 Induced from Hybridoma Cells Analysis of Anti-Aβ Monoclonal Antibody (mAb) IIA2 mAb prepared from the results of ELISA reacts with Aβ _1-42 and Aβ _1-13 peptides was gotten. However, no reaction was observed with Aβ _13-28 , Aβ _28-42 peptide and proteins not related to Aβ (Table 1). Furthermore, mAb specifically reacted with the _beads on which Aβ _1-13 , which is the N-terminal region of Aβ protein, was immobilized, but no reactivity was observed in the entire region of Aβ protein (Table 2). These results suggested that anti-Aβ monoclonal antibody IIA2 derived from hybridoma cells has specificity for 13 amino acids on the N-terminal side of Aβ protein. Furthermore, the mAb isotype was IgG1 (data not shown).

As described above, the affinity for Aβ in the brain of 80-week-old Tg 2576 mice administered with mAb and F (ab ′) ₂ prepared from mAb was examined. That is, a section of the 80-week-old Tg 2576 mouse was subjected to 10 μg / mL (A) IIA2 culture solution, (B) purified Aβ-mAb, (C) purified F (ab ′) ₂ fragment, (D) purified pFc. 'Incubated with fragment or (E) control serum. The binding was detected by reverse staining with a FITC-conjugated second antibody and microscopic observation (magnification 200 times).

As a result, a large number of plaques were fluorescently stained in (B) and (C). In (A), plaques that were fluorescently stained were clearly fewer than in (B) and (C). In (D) and (E), almost no fluorescence was observed. From this, it was revealed that the F (ab ′) ₂ fragment binds well to Aβ in the brain, like all monoclonal antibodies.

Therapeutic effect of mAb on Aβ using Tg 2576 mice
After intraperitoneal administration of 250 μg of p-Aβ-mAb, pF (ab ′) ₂ or pFc ′ once weekly to a 65-week-old Tg 2576 mouse, the mouse brain was removed at 80-week-old and Aβ in the brain was removed. I investigated. Aβ deposition was examined by immunohistochemical staining of brain tissue prepared from the left hemisphere of the brain. Microscopic observation was performed at a magnification of 200 times. As a result, a large amount of Aβ deposition was observed in the untreated mouse and the mouse administered with pFc ′, but in the mouse administered with p-Aβ-mAb or pF (ab ′) ₂ , the stained portion was Almost no Aβ deposition was observed. As a result, it was confirmed that the F (ab ′) ₂ fragment also has a therapeutic effect on Alzheimer's disease, like all antibodies.

FIG. 2A shows the result of quantification of SDS-solubilized Aβ _1-40 and Aβ _1-42 in the brain of mice treated with p-Aβ-mAb, pF (ab ′) ₂ or p-pFc using ELISA. It was. In mice treated with p-Aβ-mAb, Aβ _1-40 and Aβ _1-42 decreased 90.1% and 80.4% (p <0.01), respectively, compared to mice not treated with anything. Similarly, mice treated with pF (ab ′) ₂ showed 92.8% and 90.8% (p <0.01) decrease in Aβ _1-40 and Aβ _1-42 . However, almost no therapeutic effect was observed in mice administered with p-pFc. On the other hand, the results of insolubilized Aβ _1-40 and Aβ _1-42 treated with formic acid are as shown in FIG. 2B. Similar results to solubilized Aβ were observed. From these results, it was confirmed that the F (ab ′) ₂ fragment also has a therapeutic effect on Alzheimer's disease, like all antibodies.

Therapeutic effect of direct injection of mAb into the brain of Tg 2576 mice
10 μg of p-Aβ-mAb, pF (ab ′) ₂ or p-pFc was injected into the brain of a 60-70 week-old Tg mouse only once, and amyloid deposition was examined. When p-Aβ-mAb was administered, amyloid plaque formation decreased dramatically, but many CD11b positive cells were observed with p-Aβ-mAb. On the other hand, a similar pattern was observed after pF (ab ′) ₂ injection, but at this time, almost no CD11b positive cells were observed. p-pFc activated CD11b-positive cells despite no therapeutic effect. Furthermore, in the observation with a confocal microscope, not only CD11b-positive cells but also microglial cells were observed in the same manner (data not shown).

FIG. 3A shows the result of quantifying the effect of eliminating amyloid in the brain by the treatment of Aβ-mAb with 2% SDS-solubilized Aβ. In mice injected with p-Aβ mAb and pF (ab ′) ₂ only once in the ventricle, effects were observed in both Aβ _1-40 and Aβ _1-42 . On the other hand, in mice injected with p-pFc ′, no effect was observed in either Aβ _1-40 or Aβ _1-42 in the brain. Similar results were obtained when 75-85-week-old mice were examined (FIGS. 3B, 3C, and 3D).

Anti Aβ F (ab ') after the _second intraperitoneal administration anti Aβ F (ab') ₂ blood - The study investigated whether through the brain barrier (BBB) can be eliminated A [beta], 80-week-old AD mice The anti-Aβ fragment (0.8 mg / ml) was administered intraperitoneally. A frozen section of the brain of a mouse that had passed 4 hours after administration was prepared and fluorescently stained. For staining, anti-human Aβ _1-42 rabbit antibody, biotin-labeled anti-rabbit IgG antibody and streptavidin FITC antibody were used. As a result of fluorescent staining, it was confirmed that anti-AβF (ab ′) ₂ administered intraperitoneally was bound to Aβ in the mouse brain. Further, when the biotin-labeled anti-rabbit IgG antibody was treated with streptavidin FITC to confirm whether it was non-specific staining, no fluorescence was observed. From this, it is speculated that anti-AβF (ab ′) ₂ specifically binds to Aβ in the brain, and it is considered that anti-AβF (ab ′) ₂ passed through the blood-brain barrier (BBB).

Comparison of binding specificity of rabbit Ig and F (ab ') ₂ to anti-A.BETA.
The inhibitory effect of Aβ plaque by amyloid specific rabbit Ig was evaluated. The result of direct staining of brain sections of mice treated with F (ab ′) ₂ with Aβ-specific rabbit IgG was observed under a microscope. As a result, F (ab ′) ₂ was found to bind specifically to the Aβ plaque in the brain section. Furthermore, since this F (ab ′) ₂ antibody could not block the Aβ-specific rabbit IgG antibody, it was suggested that the epitope differs from the rabbit antiserum.

It is a microscope picture which shows the localization site | part of A (beta) in a cortex and a hippocampus. A shows an overview of Aβ plaques in the brain. Black arrows indicate Aβ plaques. The bar is 500 μm (magnification 10 times). B shows Aβ plaques in the hippocampus. The bar is 100 μm (25 times magnification). C shows Aβ plaques in the cortex. The bar is 100 μm (25 times magnification). It is a figure which shows the A (beta) density | concentration extracted by 2% SDS in the brain after treating with pA (beta) -mAb, pF (ab ') _2, or p-pFc'. It is a figure which shows the A (beta) density | concentration extracted with 70% formic acid in the brain after treating with pA (beta) -mAb, pF (ab ') _2, or p-pFc'. It is a figure which shows the A (beta) density | concentration extracted by 2% SDS in the brain after administering pA (beta) -mAb, pF (ab ') _2, or p-pFc' to the 60-70-week-old mouse | mouth intraventricularly. It is a figure which shows the A (beta) density | concentration extracted by 2% SDS in the brain after administering pA (beta) -mAb, pF (ab ') _2, or p-pFc' to a 75-85 week-old mouse | mouth in the ventricle. It is a figure which shows the A (beta) density | concentration extracted with 70% formic acid in the brain after administering pA (beta) -mAb, pF (ab ') _2, or p-pFc' to the 60-70 week-old mouse | mouth in the ventricle. It is a figure which shows the A (beta) density | concentration extracted with 70% formic acid in the brain after administering pA (beta) -mAb, pF (ab ') _2, or p-pFc' to the 75-85 week-old mouse | mouth in the ventricle.

Claims (3)

An F (ab ′) ₂ fragment of an anti-β-amyloid peptide antibody, or a fragment that is a part of the fragment and includes at least the variable region thereof, or a conjugate of the fragment, and maintains the binding ability to the β-amyloid peptide A prophylactic or therapeutic agent for Alzheimer's disease, comprising an active ingredient as an active ingredient. The preventive or therapeutic agent according to claim 1, comprising an F (ab ') ₂ fragment of an anti-β-amyloid peptide antibody as an active ingredient. The preventive or therapeutic agent according to claim 1 or 2, wherein the anti-β-amyloid peptide antibody is a monoclonal antibody.

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