Antibodies to pyroglutamate amyloid-β and uses thereof

Abstract

To provide antibodies and antigen binding fragments thereof that are useful for diagnosis, prognosis and treatment of Alzheimer's disease or other β-amyloid-related diseases, and methods of making and using the antibodies or antigen binding fragments thereof.SOLUTION: The present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having specific polypeptide sequences, wherein the antibody or antigen-binding fragment thereof specifically binds 3pE Ab, preferably human 3pE Ab.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an antibody against amyloid-β (Aβ) peptide and a treatment using the antibody. In particular, the present invention relates to the field of methods for identifying and treating amyloid-related disorders. can be used for.

[0002] (Reference to electronically submitted sequence listing) This application is filed under file name "JAB7013USPSP Sequence Listing" and filed on March 11, 2019. submitted electronically via EFS-Web as an ASCII sequence listing on the creation date of The sequence listing has a size of 76kb. The sequence listing submitted via EFS-Web is No. 60 / 633,333 filed on Oct. 23, 2006, which is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Alzheimer's disease (AD) is a progressive, severe form of mental decline. Impairment of memory, cognition, reasoning, judgment, and emotional stability, ultimately leading to death. Alzheimer's disease is a degenerative brain disorder characterized clinically by progressive loss of memory. Alzheimer's disease is a common cause of progressive mental disability (dementia) in people with Alzheimer's disease worldwide. It is recognized as a major public health problem and currently affects 5 million people in the United States alone. It is estimated that over 100,000 individuals are affected. Currently, it is incurable and However, no existing treatments effectively prevent AD or reverse its symptoms or course.

[0004] The brains of individuals with AD contain amyloid plaques, amyloid angiopathy (amyloid deposits in blood vessels), The majority of these lesions, especially amyloid tangles, are characteristic lesions of the cerebrospinal fluid (cerebrospinal fluid) and neurofibrillary tangles. Neurofibroid plaques and tangles are commonly found in several areas of the brain that are important for memory and cognitive function. Amyloid plaques and amyloid angiopathy are also seen in trisomy 21 (Down syndrome). , Hereditary Cerebral Hemorrhage with Diffuse Lewy Body Disease and Dutch Amyloidosis (HCHW To characterize the brains of individuals with AD.

[0005] The main component of amyloid plaques is the β-amyloid precursor protein. Various amyloid-beta proteins are produced by cleavage of sor protein (APP). The deposition of Aβ peptides in the brain initiates the disease cascade that leads to AD. It has been hypothesized that this is an early and necessary step in the development of Aβ. , Amyloid precursor protein and presenilin genes causing familial early-onset AD The identification of mutations in α-amyloidosis indicates that alterations in amyloid metabolism are central to the pathogenic process underlying this disease. This provides strong evidence that the event is realistic.

[0006] The amyloid-β peptide with pyroglutamate at the third residue (3pE Aβ) is 3pE Aβ is the major species accumulated in the brains of AD patients. It is present in vitellogenous and mature plaques, is metabolically stable, and plays a key role in plaque seeding (pl aque seeding) and stabilization (Cynis et al. ,Molecular Neurodegeneration,2016;11:48) Detectable amounts of 3pE Aβ have not been reported in CSF or plasma, so the target peptide These results suggest that the tides are pathology specific (DeMattos et al., uron, 2012;76:1-13). Antibodies that selectively bind to 3pE Aβ have been These may be useful in therapy. Summary of the Invention [Means for solving the problem]

[0007] As embodied and fully described, the present invention provides a method for the preparation of a medicament for the preparation of a medicament for which the third residue has pyroglutamate. Antibodies and antigen-binding fragments thereof that bind to amyloid-β (3pE Aβ), Methods for producing antibodies or antigen-binding fragments thereof that bind to US Patent 6,339,663, and US Patent 6,339,663 Assay methods using strips and methods for treating Alzheimer's disease and other β-amyloid-related diseases To treat, delay the onset of, or reverse at least one disease state or symptom The present invention also relates to use of an antibody or an antigen-binding fragment thereof of the present invention for the manufacture of a medicament for the treatment of a patient suffering from atopic dermatitis, The antibody preferentially binds Aβ peptides containing 3pE over those without 3pE. Bind to.

[0008] In certain embodiments, a. SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; b. SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively; c. SEQ ID NOs: 1, 7, 3, 8, 5, and 6, respectively; d. SEQ ID NOs: 1, 2, 3, 8, 5, and 6, respectively; e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively; f. SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively; g. SEQ ID NOs: 56, 58, 3, 4, 5, and 6, respectively; h. SEQ ID NOs: 56, 7, 3, 8, 5, and 6, respectively; i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively; j. SEQ ID NOs: 56, 7, 3, 4, 5, and 6, respectively; k. SEQ ID NOs: 1, 57, 3, 4, 5, and 6, respectively; l. SEQ ID NOs: 1, 58, 3, 4, 5, and 6, respectively; or m. having the polypeptide sequences of SEQ ID NOs: 56, 2, 3, 4, 5, and 6, respectively. , heavy complementarity determining region 1 (HCDR1), H CDR2, HCDR3, light chain complementarity determining region 1 An isolated monoclonal antibody comprising the LCDR1, LCDR2, and LCDR3 regions. or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is an antibody against 3pE Aβ, preferably An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human 3pE Aβ is described herein.

[0009] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO:9, 11, 13, 15, 16, 17, 19, 20, or 21. Heavy chain variable region having the polypeptide sequence SEQ ID NO: 10, 12, 14, 18, 22, 5 3, or 55, nothing.

[0010] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:22 a light chain variable region having a peptide sequence; b. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a polypeptide of SEQ ID NO:10 a light chain variable region having a peptide sequence; c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:11 and a polypeptide sequence of SEQ ID NO:12. a light chain variable region having a peptide sequence; d. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:13 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; e. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; f. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:16 and a polypeptide sequence of SEQ ID NO:14. a light chain variable region having a peptide sequence; g. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:20 and a polypeptide sequence of SEQ ID NO:14. a light chain variable region having a peptide sequence; h. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:17 and a polypeptide sequence of SEQ ID NO:18. a light chain variable region having a peptide sequence; i. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and a polypeptide sequence of SEQ ID NO:18 a light chain variable region having a peptide sequence; j. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:53 a light chain variable region having a peptide sequence; k. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:55. It comprises a light chain variable region having a peptide sequence.

[0011] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric. In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is human or It has been standardized.

[0012] In certain embodiments, the isolated monoclonal antibody comprises: a. a heavy chain amino acid sequence of SEQ ID NO:37 and a light chain amino acid sequence of SEQ ID NO:38; b. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:38 , c. a heavy chain amino acid sequence of SEQ ID NO: 37 and a light chain amino acid sequence of SEQ ID NO: 52; or d. a heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:54 Includes.

[0013] In certain embodiments, the antigen-binding fragments include Fv, F(ab'), F(ab')2, and s The antibody or antigen-binding fragment thereof is selected from the group consisting of 3pEAβ peptide. It selectively binds to peptides (e.g., Aβ3pE-40 and Aβ3pE-42) and other Aβ Little cross-reactivity to peptides or β-amyloid precursor protein (APP) None or none at all.

[0014] An isolated antibody encoding the monoclonal antibody or antigen-binding fragment thereof disclosed herein. Nucleic acids are also provided.

[0015] A vector comprising an isolated nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof of the present invention. A translator is also provided.

[0016] A vector comprising an isolated nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof of the present invention. Also provided is a host cell comprising the vector. Hybridomas that produce antigen-binding fragments of the antibody are also provided.

[0017] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutical and a physiologically acceptable carrier.

[0018] For the formation of plaques containing β-amyloid protein in a subject in need thereof Methods for treating related conditions are also provided. The methods include using the monoclonal antibodies or or an antigen-binding fragment thereof, or a pharmaceutical composition of the present invention to a subject in need thereof. In certain embodiments, the condition is Alzheimer's disease. The condition is dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease Hereditary encephalopathy with cerebrovascular disease, inclusion body myositis, cerebral amyloid angiopathy, and Dutch-type amyloidosis Blood (HCHWA-D).

[0019] Methods for reducing plaques associated with Alzheimer's disease in a subject in need thereof - Patent application The method also includes using a monoclonal antibody or an antigen-binding fragment thereof of the invention, comprises administering a pharmaceutical composition of the present invention to a subject in need thereof.

[0020] Methods for preventing the seeding activity of 3pE Aβ in a subject in need thereof are also provided. The method comprises using a monoclonal antibody or antigen-binding fragment thereof of the invention, or a The method includes administering the disclosed pharmaceutical composition to a subject in need thereof.

[0021] A method for producing a monoclonal antibody or antigen-binding fragment thereof of the invention, the method comprising: under conditions that produce a monoclonal antibody or an antigen-binding fragment thereof, Culturing a cell containing a nucleic acid encoding the antibody or antigen-binding fragment thereof; and recovering the bound fragment.

[0022] Methods for producing the pharmaceutical compositions of the invention are also provided. The antibody or antigen-binding fragment thereof is combined with a pharma- ceutical carrier to obtain a pharmaceutical composition. This includes:

[0023] One embodiment includes kits and devices comprising the above-described antibodies or antigen-binding fragments thereof.

[0024] Further objects, features and advantages of the present invention will become apparent from detailed consideration of the preferred embodiments which follow. It will be obvious to the trader. [Brief description of the drawings]

[0025] [Figure 1] Sensorgrams (single cycle kinetics) from surface plasmon resonance label-free detection of affinity binding interaction of BAMB31_2a (mIgG2a) to human Aβ (3pE-40) peptide. Grey traces represent double reference subtracted data, while black traces represent fitted values. [Diagram 2] 16A-C are sensograms (single cycle kinetics) of surface plasmon resonance label-free detection of affinity binding interaction of mE8c mIgG2a to human Aβ (3pE-40) peptide. The grey trace represents the double reference subtracted data, while the black trace represents the fitted values. [Figure 3A] FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3B] FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3C]FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3D] FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3E] FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3F] FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3G] FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3H] FIG. 1 shows the reactivity of BAMB674 and BAMB675 versus a control HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3I]Figure 1 shows the reactivity of BAMB674 and BAMB675 versus the reference HFA molecule to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. Results are shown for a primary antibody concentration of 0.05 μg / mL. Arrows indicate areas of plaque labeling for BAMB674 and BAMB675. (A) BAMB674, (B) BAM675, (C) Antibody I, (D) Antibody II, (E) B12L, (F) CI-C7, (G) hE8L, (H) R17L, (I) R17. [Figure 4A] FIG. 13 is a graph showing BAMB31 — 1 selectivity as demonstrated by detection of synthetic human Aβ peptides in a sandwich ELISA. [Figure 4B] Graph showing BAMB31_1 selectivity as demonstrated by detection of synthetic human Aβ peptides in a sandwich ELISA: (A) Aβ1-40 (B) AβpE11-40. [Figure 5A] FIG. 1 shows the reactivity of BAMB246 (huIgG1 chimera) to plaques by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 5B] FIG. 1 shows the reactivity of BAMB246 (huIgG1 chimera) to plaques by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 5C] FIG. 1 shows the reactivity of BAMB674 to plaques by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 5D] FIG. 1 shows the reactivity of BAMB674 to plaques by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 5E]FIG. 1 shows the reactivity of BAMB675 to plaques by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 5F] BAMB675 immunohistochemical reactivity to plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. Small inset panels show whole brain sections and enlarged areas stained. [Figure 6A] FIG. 1 shows immunohistochemical reactivity to plaques in cryopreserved AD brain tissue with 4G8 at two different magnifications. [Figure 6B] FIG. 1 shows immunohistochemical reactivity to plaques in cryopreserved AD brain tissue with BAMB31_2a (mIgG2a) at two different magnifications. [Figure 6C] FIG. 1 shows immunohistochemical reactivity to plaques in cryopreserved AD brain tissue with 4G8 at two different magnifications. [Figure 6D] FIG. 1 shows immunohistochemical reactivity to plaques in cryopreserved AD brain tissue with BAMB31_2a (mIgG2a) at two different magnifications. [Figure 7] FIG. 1 is a graph showing serum antibody concentrations at different time points following a single 20 mg / kg intraperitoneal (ip) dose in transgenic mice. [Figure 8] FIG. 13 is a graph showing microhemorrhages as assessed by number of Perl positive cells following chronic treatment with isotype control and BAMB31_2a (mIgG2a) antibodies in PDAPP mice. [Figure 9] 1 is a graph showing amyloid burden after chronic treatment with BAMB31_2a (mIgG2a) antibody in the hippocampus of isotype control and PDAPP mice as measured by an immunoassay detecting Aβ1X. Grey values ​​represent data points below the detection limit of the assay. [Figure 10] FIG. 1 is a schematic diagram of a two-compartment model for monkey PK characterization of BAMB674 and BAMB675. [Figure 11] 1 is a graph showing PK vs. observed data for BAMB674 and BAMB675. Blood concentrations of BAM31 HFA mAb as +YTE IgG1 (YTE serum) isotype and wild type IgG1 (WT serum) following intravenous (iv) bolus administration of 25 mg / kg in cynomolgus monkeys. Anti-Aβ 3pE antibody (3pE-AB) μg / mL concentration is shown on a logarithmic scale on the Y-axis over time in days on the X-axis. The calculated half-life (t1 / 2) for each mAb is shown on the inset text. [Figure 12] Graph showing brain concentrations observed for BAMB674 and BAMB675. Brain lysate levels on days 7 and 42 of BAMB31 HFA mAb as +YTE IgG1 (YTE brain) isotype and wild type IgG1 (WT brain) following iv bolus administration of 25 mg / kg in cynomolgus monkeys. Anti-Aβ 3pE antibody (3pE-AB) μg / mL concentration is shown on a logarithmic scale on the Y-axis over time in days on the X-axis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Various publications, articles and patents are cited or referenced in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which is included in the specification is indicative of the context of the present invention. Such discussion is intended to provide a basis for understanding whether any or all of these matters are disclosed or disclosed. is admitted to be part of the prior art to any claimed invention. isn't it.

[0027] The present invention is not limited to any particular methods, reagents, compounds, compositions, or biological systems. It should be understood that these are not necessarily necessarily necessarily related terms, but may be varied. The terminology used is for the purpose of describing particular embodiments only and is not intended to be limiting in any way. It should also be understood that this is not the case.

[0028] Unless otherwise defined, all technical and scientific terms used herein refer to the It has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. Certain terms used herein have the meanings set forth herein.

[0029] As used in this specification and the appended claims, the singular forms "a," "an," and " It is important to note that "the" includes multiple referents unless the context makes clear otherwise. There is.

[0030] Unless otherwise noted, the term "at least" preceding a series of elements refers to every element in the series. It should be understood that the present invention refers to elements of the It will be appreciated that the present invention is not limited to the above embodiment, and that many equivalents to the specific embodiments described herein may be recognized. Such equivalents are intended to be encompassed by this invention. can be.

[0031] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having "," "contains," or "containing," or any of these terms Other variations of the invention are intended to include the stated integer or groups of integers, but are not limited to these. is not intended to exclude any other integer or group of integers other than , and is non-exclusive or non-limiting. It will be understood that the present invention is intended to be understood as including, for example, a composition, mixture, process, or the like that includes a series of elements. The methods, articles, or apparatus are not necessarily limited to those elements expressly set forth herein. Any composition, mixture, process, method, article, or apparatus not listed in the In addition, unless expressly stated to the contrary, "or" may be used without the words "or" or "without" as the term. " refers to an inclusive "or" and not an exclusive "or." For example, A condition A or B is true (or exists) and B is false (or does not exist). if A is false (or does not exist) and B is true (or exists), then If both A and B are true (or exist), then it is satisfied by either one of.

[0032] As used herein, the conjunctive term "and / or" between multiple listed elements means Both individual and combined options are understood to be encompassing. For example, When elements are connected by "and / or," the first option may be used without the second option. The first element is applicable. The second option is that the second element can be applied without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is within the meaning and therefore the As used in the specification, it is understood that the requirement of the term "and / or" is met. The simultaneous applicability of two or more of the following is also included within the meaning, and thus the term "and / or" It is understood that the above requirements are met.

[0033] As used herein, the terms "consists of" or "consist Variations such as "of" or "consisting of" are included in the specification and claims. When used throughout the range, any enumerated integer or group of integers is inclusive, but additional Indicates that the integer or group of integers is not added to the specified method, structure, or composition.

[0034] As used herein, the term "consists essentially of" or "consist essentially of" or "consist essentially of" Variations such as "isting essentially of" are used throughout the specification and claims. When used herein, any enumerated integer or group of integers is inclusive, and optionally, the specified method, structure, or any enumerated integer or integers that do not materially change the basic or novel characteristics of the composition. Indicates inclusion of the set of integers. See MPEP §2111.03.

[0035] antibody The present invention has a particular preference for 3pE Aβ peptides over Aβ peptides that do not contain 3pE. The present invention provides an antibody or an antigen-binding fragment thereof that binds to 3pE Aβ peptide. or an antigen-binding fragment thereof, and an antibody or an antigen-binding fragment thereof that binds to 3pE Aβ peptide. The present invention further provides a method for producing a hybridoma producing an antigen-binding fragment of the Methods for treating Alzheimer's disease and other beta-amyloid-related diseases in individuals Methods for removing plaques associated with Zheimer's disease or other beta-amyloid-related diseases; and The present invention also includes a method for inhibiting the plaque-seeding activity of 3pE Aβ. The present invention provides kits and devices comprising the antibodies or antigen-binding fragments thereof for use in the methods described herein. do.

[0036] According to a particular embodiment, the present invention provides a method for producing a method for treating a pulmonary circulation comprising the steps of: a. SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; b. SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively; c. SEQ ID NOs: 1, 7, 3, 8, 5, and 6, respectively; d. SEQ ID NOs: 1, 2, 3, 8, 5, and 6, respectively; e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively; f. SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively; g. SEQ ID NOs: 56, 58, 3, 4, 5, and 6, respectively; h. SEQ ID NOs: 56, 7, 3, 8, 5, and 6, respectively; i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively; j. SEQ ID NOs: 56, 7, 3, 4, 5, and 6, respectively; k. SEQ ID NOs: 1, 57, 3, 4, 5, and 6, respectively; l. SEQ ID NOs: 1, 58, 3, 4, 5, and 6, respectively; or m. having the polypeptide sequences of SEQ ID NOs: 56, 2, 3, 4, 5, and 6, respectively. , heavy complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 An isolated monoclonal antibody comprising (LCDR1), LCDR2, and LCDR3 or an antibody thereof The antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that binds to 3pE Aβ, preferably human 3p The present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to E Aβ.

[0037] According to another particular embodiment, the present invention relates to the nucleic acid sequences of SEQ ID NOs: 9, 11, 13, 15, 16, 17, 1 9, 20, or 21. or SEQ ID NO: 10, 12, 14, 18, 22, 53, or 55 and at least 95 An isolated monoclonal antibody comprising a light chain variable region having a polypeptide sequence identical to that of Concerning antigen-binding fragments.

[0038] According to another particular aspect, the present invention comprises l. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:22 a light chain variable region having a peptide sequence; m. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a polypeptide of SEQ ID NO:10 a light chain variable region having a peptide sequence; n. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:11 and a polypeptide sequence of SEQ ID NO:12. a light chain variable region having a peptide sequence; o. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; p. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; q. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:16 and the polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; r. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:20 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; s. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:17 and a polypeptide sequence of SEQ ID NO:18 a light chain variable region having a peptide sequence; t. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and a polypeptide sequence of SEQ ID NO:18 a light chain variable region having a peptide sequence; u. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:53 a light chain variable region having a peptide sequence; v. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:55. Isolated monoclonal antibody or antigen-binding fragment thereof comprising a light chain variable region having a peptide sequence Regarding.

[0039] In one embodiment, the present invention relates to SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 58, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 2, 3, 4 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 58, 3, 4, 5, and 6, respectively. having HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has at least 85% identical sequence to SEQ ID NO:21, preferably or 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or has a heavy chain variable region having a polypeptide sequence 99% identical to SEQ ID NO: 22 or 53 or 55 and at least 85%, preferably 90%, more preferably 95% or more, e.g. 95% 96%, 97%, 98%, or 99% identical polypeptide sequence to the light chain variable region. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises the polypeptide of SEQ ID NO:21. A heavy chain variable region having a polypeptide sequence and a polypeptide of SEQ ID NO: 22 or 53 or 55. The light chain variable region has the sequence:

[0040] In one embodiment, the present invention relates to SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 58, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 2, 3, 4 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 58, 3, 4, 5, and 6, respectively. having HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has at least 85% identical sequence to SEQ ID NO: 20, preferably or 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or has a heavy chain variable region having a polypeptide sequence that is 99% identical to SEQ ID NO: 14, and 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% , 98%, or 99% identical polypeptide sequence. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:20. and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

[0041] In one embodiment, the present invention relates to SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 7, 3, 4 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3. In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has a similar sequence to SEQ ID NO:19, preferably at least 85%. Preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or a heavy chain variable region having a polypeptide sequence at least 99% identical to SEQ ID NO: 18. At least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, %, 98%, or 99% identical polypeptide sequence to the light chain variable region of the human genomic DNA. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:19. and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18. .

[0042] In one embodiment, the present invention relates to SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 7, 3, 4 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3. In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has a similar sequence to SEQ ID NO:17, preferably at least 85%. Preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or a heavy chain variable region having a polypeptide sequence at least 99% identical to SEQ ID NO: 18. At least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, %, 98%, or 99% identical polypeptide sequence to the light chain variable region of the human genomic DNA. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:17. and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18. .

[0043] In one embodiment, the present invention relates to SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 7, 3, 4 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively. having HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has at least 85% identical sequence to SEQ ID NO: 16, preferably or 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or has a heavy chain variable region having a polypeptide sequence that is 99% identical to SEQ ID NO: 14, and 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% , 98%, or 99% identical polypeptide sequence. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:16. and a light chain variable region having the polypeptide sequence of SEQ ID NO:14.

[0044] In one embodiment, the present invention relates to SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 7, 3, 4 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3. In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has a similar sequence to SEQ ID NO:15, preferably at least 85%. Preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or a heavy chain variable region having a polypeptide sequence at least 99% identical to SEQ ID NO: 14. At least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, %, 98%, or 99% identical polypeptide sequence to the light chain variable region of the human genomic DNA. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:15. and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14. .

[0045] In one embodiment, the present invention relates to SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively, or SEQ ID NOs: 56, 7, 3, 4 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3. In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has a similar sequence to SEQ ID NO:13, preferably at least 85%. Preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or a heavy chain variable region having a polypeptide sequence at least 99% identical to SEQ ID NO: 14. At least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, %, 98%, or 99% identical polypeptide sequence to the light chain variable region of the human genomic DNA. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:13. and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14. .

[0046] In one embodiment, the present invention relates to SEQ ID NOs: 1, 7, 3, 8, 5, and 6, or SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively, or SEQ ID NOs: 56, 7, 3, 8 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3. In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof has a similar sequence to SEQ ID NO:11, preferably at least 85%. Preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or a heavy chain variable region having a polypeptide sequence at least 99% identical to SEQ ID NO: 12. At least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, %, 98%, or 99% identical polypeptide sequence to the light chain variable region of the human genomic DNA. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:11. and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12. .

[0047] In one embodiment, the present invention relates to SEQ ID NOs: 1, 7, 3, 8, 5, and 6, or SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively, or SEQ ID NOs: 56, 7, 3, 8 , 5, and 6, or the polypeptide sequences of SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR 3. In another embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising: The isolated monoclonal antibody or antigen-binding fragment thereof preferably has a sequence identical to SEQ ID NO:9, at least 85%, or 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or has a heavy chain variable region having a polypeptide sequence that is 99% identical to SEQ ID NO: 10, and 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% , 98%, or 99% identical polypeptide sequence. The isolated monoclonal antibody or antigen-binding fragment thereof has the polypeptide sequence of SEQ ID NO:9. and a light chain variable region having the polypeptide sequence of SEQ ID NO:10.

[0048] In another particular aspect, the isolated monoclonal antibody comprises: a. a heavy chain amino acid sequence comprising SEQ ID NO:37 and a light chain amino acid sequence comprising SEQ ID NO:38 , b. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:38 , c. a heavy chain amino acid sequence comprising SEQ ID NO:37 and a light chain amino acid sequence comprising SEQ ID NO:52 or d. a heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:55 Includes.

[0049] In another particular aspect, the present invention provides an isolated monoclonal antibody or its antigen binding molecule of the present invention. With respect to the chimeric fragment, the antibody or antigen-binding fragment thereof is chimeric.

[0050] In another particular aspect, the present invention provides an isolated monoclonal antibody or its antigen binding molecule of the present invention. With respect to the antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof is human or humanized.

[0051] According to another particular aspect, the present invention relates to an antigen-binding fragment, which is an Fv, F( The antibody or fragment thereof is selected from the group consisting of F(ab'), F(ab')2, and scFv. The antigen-binding fragment of Aβ peptide (e.g., Aβ3pE-40 and Aβ3pE- 42) and binds selectively to other Aβ peptides or β-amyloid precursor protein (AP There is little or no cross-reactivity to P.

[0052] In another general aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof of the invention. The present invention relates to an isolated nucleic acid encoding a protein comprising the amino acid sequence of the protein. The coding sequence of a protein can be altered (e.g., substituted, deleted, or inserted). It will be understood by those skilled in the art that the amino acid sequence of a protein can be changed. A nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention without It will be understood by those skilled in the art that the sequence can be altered.

[0053] In another general aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof of the invention. The present invention relates to a vector comprising an isolated nucleic acid encoding the vector, or any vector known to those of skill in the art in light of the present disclosure, such as a viral vector. In some embodiments, the vector may be a recombinant vector such as a plasmid. A vector is a recombinant expression vector. The vector may contain, for example, a promoter, a ribosome binding element, , terminators, enhancers, selection markers, and replication origins. The promoter may include any element for establishing the function of the promoter. The promoter may be a gene-specific, inducible, or reconfigurable promoter. Numerous expression vectors are known in the art that can be used to express antibodies or their antigen binding sites in cells. Conventional cloning techniques can be used herein to generate fusion fragments. or using artificial gene synthesis to generate recombinant expression vectors according to embodiments of the invention. It is possible.

[0054] In another general aspect, the invention provides a monoclonal antibody of the invention or an antigen-binding In view of the present disclosure, the present invention relates to a host cell comprising an isolated nucleic acid encoding the fragment. Any host cell that is compatible with the recombinant expression of the antibody or antigen-binding fragment thereof of the present invention may be used. In some embodiments, the host cell is an E. coli TG1 or BL21 cell. (e.g., in the case of expression of scFv or Fab antibodies), CHO-DG44 or CHO- K1 cells, or HEK293 cells (for example, in the case of expression of full-length IgG antibodies). According to certain embodiments, the recombinant expression vector is capable of hosting a host cell such that the recombinant nucleic acid is efficiently expressed. Stably integrated into the host cell genome, by chemical transfection, heat shock, or The host cells are transformed by conventional methods such as electroporation.

[0055] In another general aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof of the invention. A method for producing a monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising: Under these conditions, cells containing nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof are and extracting the antibody or antibody complex from the cells or cell culture (e.g., from the supernatant). and recovering the expressed antibody or its antigen-binding fragment. Fragments are collected from the cells and purified according to conventional techniques known in the art and as described herein. It can be purified as described in the specification.

[0056] The present invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to 3pE Aβ. As used herein, the term "antibody" refers to an immunoglobulin capable of binding to an antigen or a portion thereof. and an immunoglobulin tag capable of specifically binding to 3pE Aβ. The binding of an antibody to an antigen can be measured by methods known to those skilled in the art. One example is the use of a BIAcore™ instrument. Generally speaking, antibodies or The antigen-binding antibody fragment has a dissociation constant of 1 μM or less, preferably 100 nM or less, and most preferably An antibody is said to specifically bind to an antigen when its binding affinity is 10 nM or less.

[0057] An antigen-binding fragment of an antibody is capable of binding to the antigen that the intact antibody binds, and is involved in antigen binding. An antigen-binding fragment refers to a fragment of an antibody that competes with the intact antibody for antigen binding. As antigen-binding fragments, they include a portion of an intact antibody that binds the antigen (i.e., the variable region of the intact antibody). includes, but is not limited to, Fab, Fab', F(ab') 2 , Fv fragment, disulfide Disulfide stabilized Fv fragment (dsFv), (dsFv)2, dual specific isomeric dsFv (dsFv-dsFv'), single chain antibody molecules (e.g., scFV), diabodies Dibodies, minibodies, nanobodies, linear antibodies, single domain antibodies (sdab), camelized single domain antibodies, multispecific antibodies formed from antibody fragments and any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. can.

[0058] Antibodies consist of two heavy chains and two light chains. Each heavy chain contains one variable domain or region. (V H ), followed by a constant domain or region (C H 1), the hinge region, and two further constant Domain or area (C H 2 and C H 3). Each light chain has one variable domain or region. Area(V L ) and one constant domain or region (C L The heavy and light chain variable domains The domain or region is a region that produces antibodies (similar to a lock) specific for a particular epitope (similar to a key). The paratope is formed by precisely binding the paratope and the epitope together. In the variable domain, the β-terminal region of each of the three variable loops in the light chain and the heavy chain is -strands are responsible for binding to antigens. These loops are called complementarity determining regions (CDRs). , i.e., CDR1, CDR2, and CDR3).

[0059] CDRs are defined as the complementarity determining regions of an antibody. These are primarily responsible for binding to the antigen. The heavy and light chain hypervariable regions of an antibody are responsible for the binding of the heavy and light chains of the antibody. There are three CDRs (CDR1, CDR2 and CDR3). The heavy chain variable complementarity determining regions are alternatively referred to as LCDR1, LCDR2, and LCDR3. The constant regions are alternatively referred to as HCDR1, HCDR2, and HCDR3. can be defined in a number of ways. For example, the CDRs in a variable region can be defined according to the Kabat, Chot hia, IMGT definitions, and / or conformational definitions, or C as known in the art Antibody CDRs may be identified according to any method of DR determination. et al., 1992, Sequences of Proteins of Imm unological interest, 5th ed.,Public Healt Originally defined by the National Institutes of Health (NIH), Washington DC The hypervariable region, Chothia (Chothia et al., Nature 34 2:877-883 (1989)) The numbering system of IMGT (Lefranc, The Immunologist 7:1 32-136 (1999); Lefranc, et al., Nucleic Aci ds Res.27:209-212(1999), Scaviner et al. Exp.Clin.Immunogenet.16:234-240(1999),Le franc,et al.,Nucleic Acids Res.43:D413-4 22(2015).

[0060] When used in the context of antibodies, "isolated" means "by the hand of man" or any means that the substance has been changed from its natural state, i.e., if it has been isolated naturally, It is either changed or removed from its environment, or both. Although naturally occurring antibodies that exist naturally in animals living in the wild are not "isolated," The same antibody that is separated from the pre-existing material in its natural state is "isolated." For example, an "isolated antibody" may refer to an antibody having a different antigen specificity. 3pE Aβ The antibody can refer to an antibody that is substantially free of antibodies that do not bind to 3pE Aβ. can occur in non-naturally occurring compositions, such as immunoassay reagents, and are When the term is used in this document, it means, within the meaning of the term, that the compound is isolated in a composition. The antibody can remain as it is.

[0061] The method of producing antibodies involves inoculating a host with the desired immunogen. mice, rats, hamsters, guinea pigs, rabbits, chickens, donkeys, horses, monkeys, Chimpanzees, orangutans, gorillas, humans, and other animals that are capable of eliciting mature immune responses Immunization procedures include, but are not limited to, any species that can be immunized. The immunoassay has been established in Edition, edited by David Wild(Nature Pub This has been described in many papers and publications, including the English Group, 2000. do.

[0062] Preferably, the immunogen embodying features of the invention is administered to a host subject, e.g., an animal or a human. The vaccine is administered in combination with an adjuvant. Suitable adjuvants include Freund's adjuvant ( Freund's adjuvant, powdered aluminum hydroxide (alum), combined with Bordetella pertussis Aluminum hydroxide and monophosphoryl lipid A - synthetic trehalose dicorynomycole Examples of suitable ion exchange media include, but are not limited to, ion exchange media.

[0063] Typically, the immunogen or combination of immunogen and adjuvant is administered by one or more subcutaneous injections. Alternatively, the mammalian host may be injected intraperitoneally. The program is carried out for at least one week, and more preferably for two weeks or more. Polyclonal antibodies produced in this manner can be produced using methods well known in the art. The protein can be isolated and purified by the above procedure.

[0064] Monoclonal antibodies are described in detail by Kohler and Milstein, for example in Nature. 256:495-497 (1975), produced by established hybridoma methods. Hybridoma methods typically involve immunizing a host or lymphocytes derived from a host. and the monoclonal antibodies secreting or potentially secreting lymphocytes. and harvesting the lymphocytes, fusing them with immortalized cells, and extracting the desired monoclonal antibodies. and selecting cells that secrete the body.

[0065] The host is immunized to produce, or is capable of producing, antibodies specific to the immunogen. Alternatively, lymphocytes can be immunized in vitro. If human cells are desired, peripheral blood lymphocytes can be used, although Alternatively, spleen cells or lymphocytes from other mammalian sources are preferred.

[0066] Lymphocytes can be fused with immortalized cell lines to form hybridoma cells. This is a process that can be facilitated by the use of a flux, e.g., polyethylene glycol. Examples include mutant rodent, bovine, or human myeloma cells immortalized by transformation. In contrast to unfused immortalized cells, the substance is hybridoma cells. Thus, after fusion, for example, hypoxanthine guanine phosphatase is preferably used. Non-fusion was achieved by using mutant myeloma cells lacking the gene for fluoribosyltransferase (HGPRT). The cells can be grown in a suitable medium that inhibits the growth or survival of the immortalized cells. In such cases, hypoxanthine, aminopterin, and thymidine were added to the medium (HAT It is added to the culture medium to prevent the growth of HGPRT-deficient cells and simultaneously inhibit the growth of hybridomas. It can allow proliferation.

[0067] Preferably, the immortalized cells fuse efficiently and are mixed and confluent by selection in a medium such as HAT. The immortalization process can be performed by isolating the antibody from a population of vectors and supporting stable, high-level expression of the antibody after fusion. Cell lines were obtained from the American Type Culture Collection, Ma Includes myeloma cell lines available from Nassas, VA.

[0068] One aspect of the present invention is to produce monoclonal antibodies that bind to amyloid β peptide. Such methods are generally known to those skilled in the art. Generally, the objectives of the present study are to (i) select a host for antibody production and (ii) provide a desired antibody to the host. and (iii) producing monoclonal antibodies that bind to the immunogen. To generate fusion cells capable of producing the desired fusion genes, a cell line derived from an inoculated host is serially divided. (iv) cloning the fused cells to produce hybridoma cell lines. and obtaining the

[0069] One method of the present invention is capable of producing monoclonal antibodies that bind to the 3pE Aβ peptide. The hybridomas are then incubated with Freund's adjuvant an initial intraperitoneal injection of a desired immunogen, such as an Aβ peptide with pyroglutamate in a naive mouse model; Then, for example, booster injections every 1-2 weeks are administered to hybrid mice such as Balb / c mice. The antibody can be produced by immunizing an animal capable of producing the antibody. Fusion of the resulting spleen can be carried out using any technique commonly known to those skilled in the art, preferably by A modified procedure of Kohler and Milstein (Eur. J. Immunol., 197 Hybridization may be performed using SP2 / 0 cells according to the method described in (6;6:292-295). The hybridomas were screened to identify hybridomas that produce antibodies specific to the 3pE Aβ peptide. Screening can be performed using standard assays such as ELISA or RIA. In one embodiment of the present invention, the monoclonal antibody BAMB3 1_1 or a method for generating a hybridoma cell line producing a humanized version thereof. .

[0070] Monoclonal antibodies can be prepared, for example, as described in U.S. Pat. No. 4,166,452. The DNA code may also be produced by recombinant gene methods known in the art. Monoclonal antibodies can be produced using conventional procedures, for example by isolating mouse heavy and light antibody chains. Antibodies that specifically bind to genes and are preferably specific for Aβ having pyroglutamate. Exploring DNA isolated from monoclonal antibody-secreting hybridoma cell lines , can be isolated and sequenced using oligonucleotide probes.

[0071] Antibody fragments which contain specific binding sites for the amyloid β peptide can also be generated. Such fragments include F(ab') which can be generated by pepsin digestion of the antibody molecule. ) 2 Fragments, and F(ab') 2 This can occur by reducing the disulfide bridges of the fragments. Alternatively, a fragment having the desired specificity may be used. To allow for rapid and easy identification of monoclonal Fab fragments, a Fab expression label was developed. A library may be constructed (Huse et al., Science 256:1270 Fab, Fv, and ScFv antibody fragments can all be grown in E. coli. These fragments can be expressed in and secreted from E. coli, allowing for the production of large amounts of these fragments. The ab'-SH fragments were directly recovered from E. coli and chemically coupled to form F(ab') 2 Cut off (Carter et al., BioTechnology 10:163-167 (1992). Other techniques for the production of antibody fragments are known to those of skill in the art. Single chain Fv fragments (scFv) are also contemplated (see, e.g., U.S. Pat. No. 5,761, (See, e.g., US Pat. Nos. 5,894 and 5,587,458.) Fv and sFv fragments contain constant regions. It is the only species with a complete combination site that does not bind to any of the ribozymes, thus reducing nonspecific binding. For example, antibody fragments are likely to exhibit reduced binding to the antibody. It may also be a "linear antibody" as described in issue 0.

[0072] Therefore, an object of the present invention is to provide an isolated monoclonal antibody expressed by the above hybridoma cell. The present invention provides a clonal antibody that specifically recognizes 3pE Aβ. The isolated monoclonal antibody may be expressed by a hybridoma cell or It can be expressed recombinantly.

[0073] Preferably, the antibody or antigen-binding fragment thereof of the present invention selectively binds to 3pE Aβ, For other Aβ or β-amyloid precursor protein (APP) that does not have 3pE Specifically, the antibody of the present invention or its antigen binding site is The combined fragments are Aβ 3pE-40 (SEQ ID NO: 40 or SEQ ID NO: 45) and Aβ 3pE-4 2 (SEQ ID NO: 51) and other Aβ peptides not containing 3pE. There is little or no cross-reactivity to tide or APP.

[0074] Table 1 provides the amino acid sequences of the antibodies of the invention. The CDRs of the heavy and light chain variable regions defined by IMGT are described as separate sequences. will be done.

[0075] [Table 1-1]

[0076] [Table 1-2]

[0077] [Table 1-3]

[0078] [Table 1-4]

[0079] [Table 1-5]

[0080] [Table 1-6]

[0081] [Table 1-7]

[0082] [Table 1-8]

[0083] [Table 1-9]

[0084] [Table 1-10]

[0085] The terms "identical" or percent "identity" refer to the identity of two or more nucleic acid or polypeptide sequences. Sequences (e.g., anti-3pE Aβ antibodies and polynucleotides encoding same, 3pE Aβ polypeptide and the 3pE Aβ polynucleotide encoding it As determined using one of the following sequence comparison algorithms or by visual inspection: are the same or unique when compared and aligned for maximum matching. Two or more sequences having a percentage of amino acid residues or nucleotides that are the same refers to a subarray.

[0086] For sequence comparison, typically one sequence serves as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer If necessary, subsequence coordinates are specified and the sequence algorithm program is executed. The sequence comparison algorithm is then run according to the designated program parameters. Based on the data, the percent sequence identity of the test sequence relative to the reference sequence is calculated.

[0087] Optimal alignment of sequences for comparison can be performed using, for example, Smith & Waterman, Adv. Appl. Math. 2:482 (1981) Local Homology Algorithm, Ne Edleman & Wunsch, J. Mol. Biol. 48:443 (1970) Same-sex alignment algorithm, Pearson & Lipman, Proc. Nat'l Similarity search method of Acad.Sci.USA85:2444 (1988), these A computerized implementation of the algorithm (Wisconsin Genetics Society ftware Package,Genetics Computer Group,5 GAP, BESTFIT at 75 Science Dr., Madison, WI , FASTA, and TFASTA), or visual inspection (typically Current Pro tocols in Molecular Biology,FMAusubel et al., eds., Current Protocols, a joint ve nature between Greene Publishing Associates es, Inc. and John Wiley & Sons, Inc., (1995 Su This can be done by means of a method such as addition (see Ausubel).

[0088] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are Altschul et al. (1990) J. Mol. Biol. 215:4, respectively. 03-410 and Altschul et al. (1997) Nucleic Aci BLAST and BLAST described in ds Res.25:3389-3402, respectively. The BLAST analysis is performed using the AST2.0 algorithm. Publicly available through the National Center for Biotechnology Information This algorithm first finds words of the same length in the database sequence. match when aligned with, or satisfy some positive threshold score T By identifying short words of length W in the query sequence, either T is the neighborhood word score threshold (Alts These initial neighborhood word hits are referred to as It serves as a seed to initiate searches to find longer HSPs that contain it. Then, work is done along each sequence for as far as the cumulative alignment score can be increased. Extends the hit in both directions.

[0089] For nucleotide sequences, the parameter M (the reward for a pair of matching residues) Core (always >0) and N (penalty score for mismatched residues, always <0) are used. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The cumulative alignment score is then reduced by an amount X from its maximum score. When the alignment is performed, the accumulation of one or more negative scoring residues results in When the cumulative score falls below zero or when the end of either sequence is reached, The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For the chido sequence, the default is word length (W) 11, expectation value 10, M=5, Use N=-4 and a comparison of both strands. For amino acid sequences, use the BLASTP program. Gram uses the default word length (W) of 3, expectation (E) of 10, and BLOSUM 62 Using the scoring matrix (Henikoff & Henikoff, Pr (see oc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0090] In addition to calculating percent sequence identity, the BLAST algorithm also Statistical analysis of similarities between columns is also performed (e.g., Karlin & Altschul, Proc. See Nat'l. Acad. Sci. USA 90:5873-5787 (1993). ). One measure of similarity provided by the BLAST algorithm is the minimum sum probability ( P(N)), which is the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid may be a nucleic acid that has a minimum match between a test nucleic acid and a reference nucleic acid. The total probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. If the sequence is less than 1, it is considered similar to the reference sequence.

[0091] Further indications that two nucleic acid sequences or polypeptides are substantially identical include the following: As described above, the polypeptide encoded by the first nucleic acid is encoded by the second nucleic acid. The objective of the present invention is to provide a method for the preparation of a polypeptide that is immunologically cross-reactive with the polypeptide to be administered. A polypeptide is typically substantially identical to a second polypeptide, e.g., two peptides Another indication that two nucleic acid sequences are substantially identical is It is when two molecules hybridize to each other under stringent conditions.

[0092] In vitro methods Assays in which the antibody or antigen-binding fragment thereof is bound to a solid phase and assays in which the antibody is in a liquid medium All formats of immunoassays using antibodies or antigen-binding fragments thereof, including certain assays, It should be understood that all such modifications are contemplated for use in accordance with the presently preferred embodiments. Antibodies embodying features of the invention can be used to detect analytes. The assay method involves the reaction of a labeled sample (analyte analogue) with the sample in a reaction with an antibody. Competitive (reagent-limited) assays and antibody-labeled single-site immunometric assays These include, but are not limited to:

[0093] The antibody or antigen-binding fragment thereof according to the present invention can be used to monitor β-amyloid-related diseases. and cell culture samples to monitor the intracellular processing of APP. Aβ3pE was detected wherever it was present, including in conditioned medium from cell cultures. Conventional immunological techniques can be used to detect the presence of the marker. Suitable immunological techniques are well known to those skilled in the art. These methods are well known and can be used, for example, by ELISA, Western blot analysis, competitive or sandwich immunoassays. These include, but are not limited to, immunoassays, all of which are well known to depend on the formation of antigen-antibody immune complexes. However, for purposes of the assay herein, the antibody or antigen-binding fragment thereof may be, for example, radioactive, enzymatic, The antibody or its antigen-binding domain may be detectably labeled with a dye, luminescent or fluorescent label, or may be a nucleic acid that binds to the antibody or antigen. The fragment can be immobilized on an insoluble carrier. Therefore, the object of the present invention is to detect A in a sample. The present invention provides an immunoassay for measuring or detecting β3pE or a fragment thereof, The method comprises the steps of: subjecting a sample having an antibody or an antigen-binding fragment thereof according to the present invention to an Aβ3pE or and contacting the antibody or antigen-binding fragment thereof with Aβ3pE or a fragment thereof to form an immune complex between the antibody or antigen-binding fragment thereof and Aβ3pE or a fragment thereof. These methods involve determining whether a cell is formed in a tissue or body fluid sample. The method can be performed in either a conventional manner, generally by obtaining a sample from the body of an individual, subjecting the sample to imaging, and by contacting the subject cells with an effective amount of a detectably labeled antibody or antigen-binding fragment thereof according to the present invention. and detecting the label to establish the presence of Aβ3pE or a fragment thereof in the sample. The method of measurement using the antibody of the present invention or an antigen-binding fragment thereof is not particularly limited. , in particular an antibody, antigen or antigen- corresponding to the amount of Aβ3pE or a fragment thereof in the solution to be measured The amount of antibody complex is detected by chemical or physical means and a standard containing a known amount of antigen is then added. Any method may be used as long as it is based on a standard curve prepared using the solutions. For example, the turbidimetric method, the competitive method, the immunoassay method, and the sandwich method are preferably used. With regard to accuracy and specificity, it is particularly preferred to use the sandwich method.

[0094] In the sandwich method, the test solution is reacted with an insoluble antibody such as an insolubilized anti-Aβ3pE antibody. The antibody is reacted with the antibody (first reaction), and then with a labeled secondary antibody (second reaction). By assaying the activity of the labeling agent on the insolubilized carrier, Aβ3pE or The amount of the fragment can be determined. The first and second reactions may be carried out simultaneously. Alternatively, the above steps may be performed continuously.

[0095] In the measurement method, labeling substances, radioisotopes, enzymes, fluorescent substances, luminescent substances, etc. are used as labeling agents. Examples of radioisotopes are: 125 1. 131 I, 3 H and 14 C is mentioned Enzymes are usually detectable by binding to an appropriate substrate which then catalyzes a detectable reaction. Examples of such enzymes include β-galactosidase, β-glucosidase, Alkaline phosphatase, peroxidase and malate dehydrogenase, preferably Examples of the luminescent substance include luminol, Luminol derivatives, luciferin, aequorin and luciferase are also included. The avidin-biotin system can also be used to label the antibodies and immunogens of the invention. When the immunogen or antibody is insolubilized, it is usually accompanied by the insolubilization or immobilization of a protein or enzyme. Either physical adsorption or chemical binding can be used for immobilization. Examples of carriers Examples of suitable polysaccharides include insoluble polysaccharides such as agarose, dextran, and cellulose; Examples include synthetic resins such as polyacrylamide and silicone polymers, and glass. can be.

[0096] In a further embodiment for detecting or diagnosing a β-amyloid related disease, the tissue, body fluid, Examples of biological samples include cerebrospinal fluid (CSF), blood, plasma, serum, urine, etc., and may be used as appropriate. The cells are then contacted with a quantity of a primary antibody to form an immune complex. The contacting is typically performed by coating the cells with the primary antibody. Contacting the sample with the primary antibody involves adding the sample to a solid matrix coated with the primary antibody. The complex formed from the elution is separated from the sample by elution. However, other recovery methods are also available. The recovered complex may be used to bind to the antigen in the complex by targeting an antigenic determinant on the antigen. The antibody is contacted with at least one secondary antibody capable of binding to an antibody against which the secondary antibody is directed. The primary determinant is the same as the antigenic determinant against which the primary antibody is directed due to the multiepitope nature of the antigen entity. Either the primary or secondary antibody may be labeled with any of the labels described above. In a preferred embodiment, the secondary antibody is detectable. The presence of detectable antibody bound to a complex consisting of the antigen bound to the second antibody is determined by the method of the art. The antibodies can be readily detected using techniques known in the art. The results were compared with those obtained with control samples to determine the presence or absence of altered Aβ3pE. The fragment levels can be measured.

[0097] In vivo methods Aspects of the present invention include methods for preventing and ameliorating amyloid-β deposition in amyloid-β-related conditions. The present invention relates to a method for treating and / or reducing the symptoms of rheumatoid arthritis, ... and / or rheumatoid arthritis, comprising administering to a subject in need thereof the methods described herein. The present invention relates to a method for treating a patient suffering from atopic dermatitis, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed in A further aspect of the invention is an antibody comprising the antibody or antigen-binding fragment thereof disclosed herein. Preventing, ameliorating, treating, and / or reducing amyloid deposition in myloid-β associated conditions The methods of the invention include pharmaceutical compositions for administering an effective amount of one or more of the compounds described herein. The method includes administering the above antibody or antigen-binding fragment thereof to a subject in need thereof.

[0098] In one aspect, the present invention relates to a method for detecting plaques containing β-amyloid protein in humans. Preventing, ameliorating, and treating amyloid-β deposition in conditions characterized by the formation of plaques The present invention relates to a method for reducing and / or preventing the onset of inflammatory bowel disease in humans, comprising administering to a human a therapeutically or prophylactically effective amount of The antibody according to the present invention or its immunologically reactive antibody, which is an antibody that specifically binds to Aβ3pE. to a human in need of such treatment, preferably peripherally. In another aspect, the present invention provides a method for inhibiting amyloid plaque formation and / or a method for inhibiting amyloid plaque formation in a human. The present invention relates to a method for removing amyloid plaques, the method comprising administering an antibody to the brain that inhibits Aβ3pE peptide. Effective amount of the present invention to sequester and induce altered Aβ3pE clearance in the brain to a human subject in need of such inhibition or removal. In an aspect, the invention provides such humanized antibodies, including their immunologically effective portions, and The present invention relates to a method for the preparation of

[0099] A subject in need thereof is at risk for developing β-amyloid plaques. The human is a human suffering from or predisposed to suffering from the condition characterized. In one embodiment, the condition is Alzheimer's disease. Down syndrome (Me21), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, or cognition associated with Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D) It is a disease.

[0100] Humanized antibodies are used to increase the similarity of antibodies to those naturally produced in humans. Humanized antibodies are antibodies derived from non-human species in which the protein sequence has been modified in a specific manner. The protein sequence is a complementarity determining factor of non-human origin that is responsible for the ability of an antibody to bind to its target antigen. The protein sequence of the human variant and the present invention are identical except for some or all of the CDR segments. The framework regions of the variable regions are qualitatively identical to the corresponding human framework regions. In some cases, the non-human CDRs are replaced by A humanized antibody is an antibody that is modified from a non-human antibody in order to retain the binding affinity and / or dissociation constant of the non-human antibody. There are minor substitutions in one or more of the CDR regions.

[0101] A humanized antibody also contains a human framework and at least one CDR from a non-human antibody. and wherein any constant region present is a human immunoglobulin constant region. Substantially identical, i.e., at least about 85%, 90%, preferably at least 95% identical or 98% identical. Therefore, all parts of the humanized antibody (one or more The CDRs of each of the sequences are substantially identical to the corresponding portions of human immunoglobulin sequences. For example, humanized immunoglobulins typically contain chimeric mouse variable region / human constant region antibodies. Does not include.

[0102] Humanized antibodies are at least three times more versatile than non-human and chimeric antibodies for use in human therapy. This has three potential advantages: 1) the effector moiety is human, making it more accessible to other parts of the human immune system; can better interact with proteins (e.g., activate microglia to promote plaque formation) 2) the human immune system does not recognize the framework or C regions of a humanized antibody as foreign Since the host would not recognize the antigen, the antibody response to such an administered antibody would be entirely foreign and non-human. 3) the effect of the administered non-human IgG antibody on the immune response should be lower than that of the administered non-human IgG antibody or partially foreign chimeric antibody. Human antibodies have been reported to have a shorter half-life in the human circulation than human antibodies. is.

[0103] Treating or preventing conditions characterized by the formation of plaques containing β-amyloid protein In the method for the treatment of cancer, the antibody or antigen-binding fragment thereof of the present invention (including an immunologically reactive fragment) is used. (including those with clinical or pre-clinical Alzheimer's disease, dementia associated with Down's syndrome, or are at risk for amyloid-β-related conditions or pathologies, such as clinical or preclinical amyloid angiopathy The drug is administered to individuals who are at risk or exhibit these conditions using standard administration techniques. is administered peripherally (i.e., not into the central nervous system), intravenously, intraperitoneally, subcutaneously, or pulmonary The antibody or conjugate thereof may be administered intradermally, intramuscularly, intranasally, buccally, sublingually, or by suppository administration. The fragments may be administered directly to the ventricular system, spinal fluid, or brain parenchyma, and techniques for targeting these locations are Although well known in the art, it is not necessary to utilize these more complicated techniques. The antibody or binding fragment thereof is effective when administered by a simpler technique that relies on the peripheral circulation. The benefits of the present invention are not provided directly to the central nervous system itself, but rather to antibodies or The antigen-binding fragment includes the ability to exert its beneficial effect.

[0104] The pharmaceutical composition for administration may be designed to suit the selected mode of administration and may contain dispersing agents, softening agents, and / or other additives. Pharmaceutically acceptable excipients such as buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers, etc. are used where appropriate. Remington's s Pharmaceutical Sciences,Mack Publishing Co., Easton Pa., latest edition is generally used by the skilled artisan. An overview of formulation techniques as known is provided.

[0105] For example, by encapsulating them in liposomes or by blocking polar groups. Thus, modifying the solubility properties of the antibodies of the invention by making them more lipophilic is particularly Could be useful.

[0106] Peripheral systemic delivery by intravenous, intraperitoneal or subcutaneous injection is preferred. The preferred vehicle for administration is simple. However, administration may also be by nasal aerosol or suppository. Drugs may also be administered through the mucous membrane. Formulations suitable for such administration are well known and include Typically, the surfactants used are those that promote intermembrane transfer. In this case, it is derived from steroids or N-[1-(2,3-dioleoyl)propyl-N ,N,N-trimethylammonium chloride (DOTMA), or Various compounds such as cholesterol hemisuccinate and phosphatidylglycerol. do.

[0107] The concentration of the humanized antibody in the formulation may range from as little as about 0.1% to about 15 or 20% by weight, primarily in the eluent. The dosage will be selected according to the particular mode of administration chosen, based on body volume, viscosity, etc. A typical pharmaceutical composition for injection is 1 mL of sterile buffered water, 1 to 1 mL of phosphate buffered saline. The formulation may be prepared so as to contain 100 mg of the humanized antibody of the present invention. It may subsequently be sterile filtered or otherwise rendered microbiologically acceptable. A typical composition for intravenous infusion would be 250 mL of fluid, e.g., sterile Ringer's solution, The antibody concentration per mL may be 1 to 100 mg / mL or more.

[0108] The antibody is administered at a dose of about 0.0001 to 100 mg / kg of the host's body weight. The dosage is in the range of 0.01 to 75 mg / kg, preferably 0.01 to 75 mg / kg. For example, the dosage is 0.02mg / kg, 0.25mg / kg, 0.5mg / kg, 0.75mg / kg g / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 10mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, 20mg / kg, 35mg / kg, 40mg / kg, 45mg / kg, 50mg / kg, 55mg / kg, It may be 60 mg / kg, 65 mg / kg, 70 mg / kg, or 75 mg / kg. In an embodiment, the dosage is within the range of 0.01 to 10 mg / kg, or 0.1 to 15 mg / kg. / kg, or within the range of 0.1 to 20 mg / kg, or within the range of 0.1 to 30 mg / kg Within the range, or within the range of 0.1 to 40 mg / kg, or within the range of 0.1 to 50 mg / kg Within the range of 0.1 to 60 mg / kg, preferably at least 1 mg / kg, at least 5mg / kg, at least 10mg / kg, at least 20mg / kg, at least At least 30 mg / kg, at least 40 mg / kg, at least 50 mg / kg, or In a preferred embodiment, the dosage is about 10 mg / kg, about 20 mg / kg, or about 60 mg / kg. g / mg, approx. 30kg / mg, approx. 40mg / kg, approx. 50mg / kg, approx. 60mg / kg or about 70 mg / kg. In particularly preferred embodiments, the antibody is administered at a dose of about 0.3 mg / kg. In an exemplary treatment regimen, the antibody is administered intraperitoneally at a dose range of 100 mg / kg to about 60 mg / kg. The body is about 10kg / mg, about 20kg / mg, about 30kg / mg, about 40mg / kg, about It is administered intraperitoneally at a dose of 50 mg / kg, or about 60 mg / kg.

[0109] As used herein, the term "about" when used in reference to a measurable value, such as an amount, , ±20% to ±0.1%, preferably ±15% or ±10%, more preferably ±5%, and Preferably, the variation is ±1%, more preferably ±0.5%, and even more preferably ±0.1%. Such variations of ±0.05% or ±0.01% from the specified value are , is appropriate.

[0110] Exemplary treatment regimens include administration once every two weeks, once a month, or once every 3 to 6 months. In some methods, two or more monoclonal antibodies with different binding specificities are They are administered simultaneously, with the dosage of each antibody administered being within the ranges indicated. The body is usually administered multiple times. The intervals between doses can be weekly, monthly, or The intervals can be annual. The intervals can also be irregular, which may increase resistance to Aβ in the subject. The antibody may be administered as a sustained release formulation, or by measuring the blood levels of the antibody. The dosage and frequency of administration can be determined based on the patient's individual needs. The half-life of the antibody in the human subject varies according to the subject's half-life. Generally, human antibodies exhibit the longest half-life, followed by The order is humanized antibodies, chimeric antibodies, and non-human antibodies.

[0111] The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or curative. In prophylactic use, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives. The progression of the disease is reduced or halted, preferably until the subject experiences partial or complete remission of symptoms of the disease. Relatively high doses may be required at relatively short intervals until a response is seen. Dimen may be administered.

[0112] In some methods, the dose is adjusted to achieve a plasma antibody concentration of approximately 1-1000 μg / mL. In some cases, it is administered at a concentration of about 25 to 300 μg / mL. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the antibody in the subject.

[0113] Treatment with the antibody of the present invention may be a sole treatment. The treatment may be one of a combination therapy regimen, in which one or more additional therapeutic agents are also used to treat the individual. It may be a component or a step.

[0114] When used for in vivo therapy, the antibodies or antigen-binding fragments thereof of the invention are administered in a therapeutically effective amount, For example, reducing, removing, or preventing β-amyloid plaques, or treating AD or The present invention relates to a method for treating cognitive impairment in a subject with a β-amyloid-related disease, the method comprising administering the method to an individual in an amount that improves cognitive function in a subject with a β-amyloid-related disease. The antibody or antigen-binding fragment thereof may be administered intravenously, e.g., as a bolus or over a period of time. Continuous injection over a range of 100 mg / kg / day can be used to treat a wide range of conditions, including intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, and intrathecal. The compositions of the present invention are administered to an individual in accordance with known methods, such as by oral, topical or inhalation routes. The agent optionally has other properties that are at least partially effective in treating an amyloidogenic disease. It can be administered in combination with drugs such as cerebrospinal fluid (CEF) and cerebrospinal fluid (CF) to treat Alzheimer's disease and the formation of amyloid deposits in the brain. In the case of related conditions occurring, the antibodies or antigen-binding fragments thereof of the invention act to bind to the antibodies of the invention across the blood-brain barrier. It may be administered in combination with other agents that increase the passage of the specific agent.

[0115] In one embodiment of the invention, the antibody or antigen-binding fragment thereof of the invention is Binds to 3pE Aβ. By binding to 3pE Aβ in plaque deposits, the antibody or an antigen-binding fragment thereof can induce plaque removal. The development of Aβ is thought to be due to activation of microglia around the plaques and the suppression of plaque function by removing stable Aβ forms. Furthermore, the antibody or antigen-binding fragment thereof of the present invention may be 3pE may prevent the plaque-seeding activity of Aβ. The possible enrichment of 3pE Aβ in the IL-1 protein may increase the therapeutic safety margin for immunotherapy. It is possible.

[0116] Kits and Devices The present invention provides kits and devices that can be used in the above-mentioned methods. Alternatively, the kit and device may comprise an antibody or an antigen-binding fragment thereof that binds to 3pE Aβ. In addition, the kit may include reagents and instructions. The instructions may be, for example, printed on paper. The instructions for use may be provided in a form and / or electronically readable medium. The information may be obtained, for example, from an internet website designated by the manufacturer or distributor of the kit. The information may be provided by directing the user to a website.

[0117] The reagents contained in the kit of the present invention are substantially adsorbed or changed by the material of the container. All kinds of containers are used so that the activity of the various ingredients is not substantially lost while the may be provided.

[0118] In one embodiment, the kit or device comprises an antibody or antigen-binding fragment thereof of the invention, preferably Preferably, the antibody is a purified antibody, more preferably a monoclonal antibody, even more preferably a 3p The present invention includes an isolated monoclonal antibody that binds to Aβ peptide. In an embodiment, the antibody , expressed by hybridoma cells.

[0119] Embodiment In embodiment 1, a. SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; b. SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively; c. SEQ ID NOs: 1, 7, 3, 8, 5, and 6, respectively; d. SEQ ID NOs: 1, 2, 3, 8, 5, and 6, respectively; e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively; f. SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively; g. SEQ ID NOs: 56, 58, 3, 4, 5, and 6, respectively; h. SEQ ID NOs: 56, 7, 3, 8, 5, and 6, respectively; i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively; j. SEQ ID NOs: 56, 7, 3, 4, 5, and 6, respectively; k. SEQ ID NOs: 1, 57, 3, 4, 5, and 6, respectively; l. SEQ ID NOs: 1, 58, 3, 4, 5, and 6, respectively; or m. having the polypeptide sequences of SEQ ID NOs: 56, 2, 3, 4, 5, and 6, respectively. , heavy complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 An isolated monoclonal antibody comprising (LCDR1), LCDR2, and LCDR3 or an antibody thereof The antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that binds to 3pE Aβ, preferably human 3p An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to E Aβ.

[0120] Embodiment 2 is SEQ ID NO: 9, 11, 13, 15, 16, 17, 19, 20, or 2 1, or a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 10. , 12, 14, 18, 22, 53, or 55. 2. The isolated monoclonal antibody of embodiment 1 or its equivalent, comprising a light chain variable region having the sequence It is an antigen-binding fragment.

[0121] In embodiment 3, a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:22 a light chain variable region having a peptide sequence; b. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a polypeptide of SEQ ID NO:10 a light chain variable region having a peptide sequence; c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:11 and a polypeptide sequence of SEQ ID NO:12. a light chain variable region having a peptide sequence; d. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:13 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; e. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; f. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:16 and a polypeptide sequence of SEQ ID NO:14. a light chain variable region having a peptide sequence; g. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:20 and a polypeptide sequence of SEQ ID NO:14. a light chain variable region having a peptide sequence; h. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:17 and a polypeptide sequence of SEQ ID NO:18. a light chain variable region having a peptide sequence; i. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and a polypeptide sequence of SEQ ID NO:18 a light chain variable region having a peptide sequence; j. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:53 a light chain variable region having a peptide sequence; k. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:55. 2. The isolated monoclonal antibody of embodiment 1, comprising a light chain variable region having a peptide sequence. or an antigen-binding fragment thereof.

[0122] Embodiment 4 is an embodiment in which the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of aspects 1 to 3.

[0123] Embodiment 5 is a method for treating an isolated monoclonal antibody or antigen-binding fragment thereof, comprising administering to a subject an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment being human or humanized. The isolated monoclonal antibody or its antibody according to any one of embodiments 1 to 4, It is the original binding fragment.

[0124] In embodiment 6, a. a heavy chain amino acid sequence comprising SEQ ID NO:37 and a light chain amino acid sequence comprising SEQ ID NO:38 , b. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:38 , c. a heavy chain amino acid sequence comprising SEQ ID NO:37 and a light chain amino acid sequence comprising SEQ ID NO:52 or d. a heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:54 The present invention is an isolated monoclonal antibody comprising:

[0125] Embodiment 7 is a method for producing a monoclonal antibody or an antibody thereof according to any one of embodiments 1 to 6. An isolated nucleic acid encoding the original binding fragment.

[0126] Embodiment 8 is a vector comprising the isolated nucleic acid of embodiment 7.

[0127] Embodiment 9 is a host cell comprising the vector of embodiment 8.

[0128] Embodiment 10 is an isolated monoclonal antibody according to any one of embodiments 1 to 6, or and a pharmaceutical composition comprising the antigen-binding fragment and a pharma- ceutical acceptable carrier.

[0129] Embodiment 11 is a method for treating a subject in need thereof, comprising administering to the subject a β-amyloid protein. A method for treating a condition associated with plaque formation, the method comprising any one of embodiments 1 to 6. 10. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, The method comprises administering the pharmaceutical composition described above to a subject in need thereof.

[0130] Embodiment 12 is the method of embodiment 11, wherein the condition is Alzheimer's disease.

[0131] Embodiment 13 is a method for treating a condition comprising administering to a patient a therapeutically effective amount of the ... associated with idiopathic Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and Dutch amyloidosis 12. The method of embodiment 11, wherein the method is selected from the group consisting of Hereditary Cerebral Hemorrhage and Asthma (HCHWA-D). It is.

[0132] Embodiment 14 relates to a method for treating plaques associated with Alzheimer's disease in a subject in need thereof. The method for reducing the amount of a monochromator according to any one of embodiments 1 to 6, 10. A method for treating a patient in need of the present invention, comprising administering to said patient a monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutical composition according to embodiment 10. The method includes administering to a subject.

[0133] Embodiment 15 is directed to preventing the seeding activity of 3pE Aβ in a subject in need thereof. A method for inhibiting a cellular function of a subject, the method comprising administering to the subject a monoclonal antibody according to any one of embodiments 1 to 6. 11. A subject in need thereof, The method comprises administering to

[0134] Embodiment 16 is a method for producing a monoclonal antibody or a method for producing a monoclonal antibody thereof according to any one of embodiments 1 to 6. A method for producing an antigen-binding fragment, comprising producing a monoclonal antibody or an antigen-binding fragment thereof. The cells containing nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof are cultured under conditions and recovering the antibody or antigen-binding fragment thereof. .

[0135] Embodiment 17 is a method for producing a monoclonal antibody or a method for producing a monoclonal antibody thereof according to any one of embodiments 1 to 6. A method for producing a pharmaceutical composition comprising an antigen-binding fragment, the method comprising: and combining the antigen-binding fragment with a pharma- ceutical acceptable carrier to obtain a pharmaceutical composition. The present invention relates to a method for EXAMPLES

[0136] The invention can be further understood by consideration of the following non-limiting examples.

[0137] Example 1: Monoclonal antibody production and humanization process Three Balb / c mice (Janvier Labs) were cultured with complete Freund's ajuva H2N-pEFRHDSGC-CO in 100% ethyl acetate (Sigma, St. Louis, MO) The primary immunization was with COOH-terminal cysteine ​​(Eurogentec) (SEQ ID NO: 47). Imject Maleimide A via the in-residue according to the manufacturer's instructions. Commercially available products include ctivated BSA kits (Pierce, Rockford, IL). Maleimide-activated bovine serum albumin (Life Technologies, Inc.) was synthesized using a commercially available kit. The peptide was synthesized by coupling to α-glucose esters of α-glucose esters (Phenologies, Carlsbad, CA). Mice were first incubated with Freund's complete adjuvant, followed by incomplete Freund's adjuvant. Coupled to 100 μg or 200 μg of BSA in Roydt's adjuvant (Sigma) The mice were boosted with the peptide every two weeks.

[0138] Hybridoma and antibody production: Mice showing the highest serum titers are selected for fusion. On the fourth day, the spleen of each mouse was isolated and frozen in liquid nitrogen. Prior to excision, all mice were cultured with BSA (Merck (Kenilworth) 100 μg of H2N-pEFRHDSGC-COOH ( The mice were boosted intraperitoneally with IgG1 (SEQ ID NO: 47). Mice spleen cells were cultured using the method of Kohler and Mils The procedure was modified from that of tein (Euro. J. Immunol., 1976; 292-295). The hybridization was performed with SP2 / 0 cells (ATCC, Manassas, VA). Domas were seeded into 30x96-well plates and after 10 days, 0.5 μg / well Uncoupled Aβ 3pE-40 peptide (AnaSpec (Fremon t, CA)) and screened by direct ELISA. Positive cells were counted with 0.5 μg / mL (Lack of) cross-reactivity with coated Aβ1-40 peptide (AnaSpec) The clones were tested and immediately subcloned.

[0139] After fusion, 17 clones were cloned using human Aβ3pE-40 synthetic peptide (SEQ ID NO: 40). The direct coated ELISA screen showed a positive reaction and the liquid Freeze in nitrogen.

[0140] All hybridomas were cultured in 10% fetal bovine serum (Hyclone (Europe)), Hybridoma Fusion Cloning Supplement(2%)( Roche (Brussels, Belgium)), 2%HT (Sigma), 1mM Sodium pyruvate, 2 mM L-glutamine and penicillin (100 U / mL) and Cultured in Dulbecco's modified Eagle's medium supplemented with streptomycin (50 mg / mL). All products were commercially available and purchased from Life Technologies. Humidified with 8% CO 2 The cells were incubated in an air incubator.

[0141] Direct ELISA for antibody selection: The staining agent used to detect the Aβ 3pE-40 antibody described above was The cleaning ELISA was performed using 0.5 μg / mL of free human Aβ 3pE-40 peptide ( SEQ ID NO: 40) overnight at 4° C. in 50 μL / well of coating buffer (10 mM T ris, 10 mM NaCl and 10 mM NaN 3 , pH 8.5) xisorp (Life Technologies) flat bottom high coupling 96 well microtaper It was a direct ELISA coated onto Iter plates.

[0142] The next day, 75 μL / well of 0.1% casein (Merck) in PBS was incubated at room temperature for 60 min. The plate was blocked with 50 μL of hybridization buffer to reduce non-specific binding. The supernatant of the doma was added and incubated at 37°C for 1 hour. After washing, the bound monoclonal antibody was The null antibodies were incubated at 37°C for 1 h in 50 μL / well of horseradish peroxidase (Ame) rsham-Pharmacia Biotech(Little Chalfont, Detection was with sheep anti-mouse IgG conjugated with IgG (United Kingdom). The reagent was diluted in 0.1% casein / PBS. The plate was washed and 50 μL of 0.42 mM 3,5,3',5'-tetramethyl-benzidine (Biorad), 0.003% (Capacity / Capacity) H 2 O 2 (Biorad) (100 mM citric acid (Biorad (H Hercules, CA), 100 mM disodium hydrogen phosphate (pH 4.3) ( A solution of 100 mM NaCl (BioRad) was added as substrate. The mixture was incubated at room temperature for up to 15 min on a plate shaker. Allow the reaction to proceed for 1 h, then add 50 μL / well of 2N HO 2 SO 4 (Merck) The reaction was stopped and the plate was read at 450 nm in a microtiter plate reader. (Thermomax, Molecular Devices). Selected monoclonal Cross-reactivity of the antibody with full-sized human free Aβ 1-40 was evaluated in the screening assay. The test was performed using the same direct ELISA as in (a).

[0143] From the 17 Aβ3pE-40-reactive clones, further selection was performed based on affinity and selectivity. BAMB31_1 was selected for characterization (see Examples 2 and 3). was determined to have a mouse IgG1 isotype heavy chain and a mouse kappa light chain. The IgG1 Fc shares 70% sequence identity and 76% sequence identity with mouse IgG2a Fc. Although they share similarities, these isotypes have distinct activities and protein profiles. Compared to mouse IgG2a, mouse IgG1 has mouse FcγRI, Fc γRIII and FcγRIV receptors and weaker binding to mouse C1q. It has weak Fc effector and complement functions. It is the isotype that most closely resembles human IgG1 activity. Therefore, mouse IgG2a is thought to bind to mouse FcγRI, FcγRIII, and Fc Clearance of Aβ plaques by binding to the γRIV receptor and mouse C1q Complement and antibody-dependent cellular cytotoxicity, which may contribute to ty, ADCC), and antibody-dependent cellular phagocytosis (antibody-dependent cellular phagocytosis). is, ADCP) activity.

[0144] The sequence of the BAMB31 heavy chain was changed from mouse IgG1 to mouse IgG2a, The preservation of reactivity after V-region cloning was confirmed by SPR analysis as described below. Confirmed by law.

[0145] Humanization process: For this work, we used the AbM definition (Martin, AC, PNAS8 6:9268-9277, 1989), except for CDR-H2, which was delineated according to Using a procedure similar to that used by Ngh, et. al. (MAbs 2015; 7(4): 778-91) The parent antibody BAMB31_2a (mIgG2a) was humanized using the complementary The CDRs were identified in the mouse parental sequence. These sequences were then compared to the human germline and and four human germline heavy chains, selecting two human germline light chain frameworks. The mouse CDRs were grafted into the framework. Mouse J segment sequence and human J segment By comparing the sequences, human J segments for the VL and VH of each parent antibody were selected. The molecular model of the Fv region of the parent mAb was constructed using default parameters. The data was generated at MOE (CCG, Montreal, Canada) using the The resulting model is examined graphically to identify potential important factors for binding and / or antibody stability. These positions were then used to identify the human / We created an antibody library with a binary combination of mouse Each chain containing the mouse CDRs was paired with the opposite mouse parental chain. Only the first chain was fitted to the human framework and backmutations were determined based on antigen binding. The humanized VH and VL were then combined to arrive at the final antibody candidate.

[0146] Library clones were expressed as Fab in E. coli and compared to the intact mouse parent molecule. The peptide binding was tested by signal transduction and ELISA. Molecular signals showing binding of more than 100% were selected for sequencing. A monoclonal human IgG1 was produced by selecting and combining a human-adapted heavy chain and a human-adapted light chain. All VH / VL humanized pairs of each antibody were expressed, purified, and expressed as antibodies. In this case, Epivax in silico immunogenetic risk, reversion to mouse sequence The number of residues that were altered and their biophysical properties were evaluated.

[0147] Result: Some residues had to be backmutated to the mouse sequence to preserve parental binding. Retention of binding, immunogenetic risk of Epivax in silico, and biological The physical properties were independent of the number of back-mutations to the mouse sequence, but their positions were similar to those of the mouse sequence. Representative HFA mAb characterization results from this analysis are shown in Tables 2 and 3. As shown in.

[0148] [Table 2] BAMB246 parent human IgG1 chimera Values ​​outside the desired range are in bold. Total number of residues backmutated to mouse sequence >= 5 , Hc and Lc Epivax risk score >-10, V domain synthetic Epivax risk score Core > -20, SEC% monomer value < 95% kd(1 / s) value > 1.00E-04, KD (M) value>2.50E-11, Tonset(℃)<60, Tm1(℃)<65, Tag g(℃)<65

[0149] Reduced risk of post-translational modifications: Parent antibody and human framework-adapted variants have HCDR2 To address this potential issue, To achieve this, we used condensation oligos to create individual libraries for both N and G residues. These new sequences were created by randomly selecting all 20 amino acids for each position. Each library was screened for retained binding. Mutant antibodies that showed similar binding to the parent were selected for sequencing.

[0150] Results: After sequencing, both the NS and NG mutants had binding comparable to the parent. The lead HFA mutant was cloned and expressed as wild-type IgG1 (BAMAB674). The antibody expressed IgG1 (Hc constant) with M37Y, S39T, and T41E point mutations in the Fc region. Constant region Fc IgG1 heavy chain constant region sequence Based on GenBank accession number AEV43323 The mutant (numbered 0-1) was named +YTE IgG1 (BAMB675). It is known to increase affinity for cRn and increase circulating half-life (Pro parties of Human IgG1s Engineered for En hanced Binding to the Neonatal Fc Recept or (FcRn), Dall'Acqua WF., JBC, 2006). BAMB6 The characterization of 74 and BAMB675 is shown in Table 3.

[0151] [Table 3]

[0152] [Table 4] * N=1

[0153] Example 2: Thermal stability test The thermostability of BAMB31 human framework-adapted mutants was demonstrated by nanoparticle analysis. Using nano differential scanning fluorimetry (NanoDSF) The melting onset temperature (Tonset), the first melting transition temperature (Tm1), and the first The temperature at which the first stage of aggregation was detected (Tagg) was measured. The data for some of the mutants are shown in Table 2 (columns 6 to 8). and shown in Table 3 (rows 10 to 12) and Table 3a.

[0154] Materials and Methods: Thermal stability of samples was determined using an automated Prometheus instrument. The measurement is performed by loading the samples from a 384-well sample plate into 24-well capillary tubes. Each sample is measured twice. Use the DSF user interface (Melting Scan tab) to Set the experimental parameters for the thermal scan of a typical IgG sample at a rate of 1.0°C / min. Temperatures range from 20°C to 95°C. Typical concentrations of samples range from 0.3 to 1 mg / mL. 3 Using molecular intrinsic fluorescence at 30 and 350 nm, amphotericin was detected during a temperature ramp. The adhesion is monitored and recorded as the change in fluorescence intensity over time. This is the result of a thermal scan. In parallel, using back reflection technology, the instrument is called the thermal run The NanoDSF method calculates the onset of aggregation (Tagg) in the pool. The conformation of the lead candidate is often monitored as an indicator of long-term sample stability under and colloidal stability can be measured simultaneously.

[0155] Example 3: Epivax in silico immunogenetic risk assessment EpiMatrix software for predicting MHC class II binding (EpiV Axox Inc. was used to perform in silico characterization of the V region of the anti-Aβ 3pE mab. The software examines consecutive amino acid 9-mers to identify potential HLA classifiers. The database identifies the most common HII binding sequences, which represent approximately 95% of the human population. When the HLA receptor of an antigen-presenting cell binds to an agretope of a peptide, In this case, the other side of the peptide (epitope) binds to T effector cells or T regulatory cells. This can stimulate an immune response against a protein bearing that epitope. The software may provide a predictive T regulatory cell binding assay to determine whether the T regulatory cell is capable of binding to the target T cell. The score is calculated based on the total number of agretopes in the protein. The results are normalized to the size and number of binding events leading to an output indicating the predicted immunogenicity of the protein. can be.

[0156] Example 4: Analytical properties of purified mAbs The protein concentration of each purified mAb was measured using a NanoDrop1000 spectrophotometer or a T Absorbance at 280 nm on a rinean DropSense 96 multichannel spectrophotometer The intensity was measured and calculated using the attenuation coefficient based on the amino acid sequence.

[0157] 0.2MN at 1mL / min for 20 min on a Waters Alliance HPLC a Phosphate pH 6.8, TOSOH TSKgel BioAs sist antibody purification by running the sample on a G3SWxl column HPLC was performed. The column eluate was monitored by absorbance at 280 nm. Results are shown in Tables 2 and 3.

[0158] Example 5: Measurement of binding kinetics and affinity Surface Plasmon Resonance (SPR) is a method for measuring biomolecular interactions. This is a label-free detection method used to investigate small changes in mass on the sensor surface. This direct real-time binding assay monitors and provides qualitative information about biomolecular interactions. provides quantitative and objective data; i.e., equilibrium binding constants (affinity, K D ) and kinetic constant (k a / k d ; complex association rate k a , and the dissociation rate of the complex k d ) is determined. The method involves the study of protein-protein and protein-nucleic acid interactions, as well as protein-small This is useful in studying the interaction of 3pE-specific antibodies with human Aβ3. The interaction between pE-40 (SEQ ID NO: 40) or Aβ3pE-28 (SEQ ID NO: 42), and human Aβ1-40 (SEQ ID NO: 41) or Aβ1-28 (SEQ ID NO: 43) and rodent A The interaction between the β3pE-28 peptide (SEQ ID NOs: 45 and 46) was investigated.

[0159] Materials and Methods SPR: A mouse antibody capture kit from GE Healthcare was used to capture Aβ-3pE-4 Affinity test of BAMB31_2a (mIgG2a) for 0 peptide (SEQ ID NO: 40) (described in U.S. Patent Application Publication No. 2018 / 0142011) RD / Aβ / pE3 / 1 mIgG2a and (U.S. Pat. No. 9,944,696 and U.S. Pat. No. mE8c mIgG2a (described in US Pat. No. 8,679,498) was included as a control antibody. Antibody-specific antibodies were assayed via amine coupling on a CM5 sensor chip according to the manufacturer's protocol. Next, the antibody of interest (1 μg / mL) was immobilized by anti-mouse antibody. After capture to a concentration of 300 RU, running buffer (2.7 mM KCl , 137 mM NaCl and 0.05% surfactant P20 (Tween™ 20) The 20 mM phosphate buffer solution was diluted with various concentrations (3.125 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM) of human Aβ 3pE-40 peptide (SEQ ID NO:4 0) was injected. The surface was washed with 10 mM glycine HCl, pH 1.7 for at least 180 seconds. The human Aβ(1-40) peptide (SEQ ID NO: 41) was used as a negative control. and used it.

[0160] Affinity measurements were performed using the optical biosensor T200 (Biacore®). Biacore T200 Evaluation Software (Version Kinetic analysis was performed using the ELISA kit (version 2.0) according to a 1:1 binding fitting model.

[0161] In some cases, the binding affinity and specificity of the anti-Aβ 3pE mAb was determined using different instruments (B Biacore T200, Biacore 8K or MASS-2 (Biacore,I nc.)) and anti-human or anti-mouse immunoglobulin biosensor surfaces. Anti-human or anti-mouse immunoglobulin antibodies were attached to the amine coupling reagent, SPR. Using the manufacturer's instructions, prepare a CM4 sensor chip or a CM5 sensor chip. The antibody of interest was covalently bound to the surface of a chip (GE Healthcare). Or capture mouse immunoglobulin on a sensor chip to a concentration of 300-400 RU, Aβ peptide or protein (e.g., human Aβ 3pE-40 (SEQ ID NO: 40), Aβ 3 pE-28 (SEQ ID NO: 42), scrambled Aβ 3pE-28 (SEQ ID NO: 44), Aβ 1-28 (SEQ ID NO: 43), mouse Aβ 3pE-28 (SEQ ID NO: 46), or fibrinogen Lonectin), containing 0.005% surfactant P20 (Tween™ 20) Inject at various concentrations in HEPES-buffered saline. 30 μL at 100 μL / min. The surface was regenerated by injecting two pulses of 10 mM Gly pH 1.5. The difference in SPR signal between flow cells containing the antibody and reference cells without the captured antibody. The data for the blank injection are taken from the reference-subtracted signal. By subtraction, the additional contribution of the instrument to the signal was removed. In this case, Biaevaluation software (Biacore, Inc.) was used. The association and dissociation phases at all concentrations (global fit) were fitted to a 1:1 binding model. Data were analyzed by combining the data. If not applicable, data were combined. Qualitative assessment was made as to whether binding was present or absent.

[0162] Immunohistochemistry of formalin-fixed, paraffin-embedded brain: To achieve this, sections were deparaffinized, rehydrated, and then transpired in formic acid (70% in distilled water) for 10 min. Antigen retrieval was performed by incubating brain slides from transgenic mice. Intrinsic peroxidase activity was measured using 3% hydrogen peroxide (DAKO; Glostrup, Dennis, MA, USA). Blocking was performed with background reduction component (DAKO, S3022) in different concentrations in the antibody diluent (working concentrations: 2, 0.1-0.05-0 .025μg / mL), BAMB674, BAMB675, hE8L, R17L, R1 7, CI-C7, B12L, Antibody I or Antibody II (the last seven antibodies are disclosed in U.S. Pat. No. 9,999,103,213). No. 44696(B2) and U.S. Pat. No. 8,679,498(B2) as previously described. After extensive washing, sections were incubated with HRP-conjugated anti-human secondary antibody (H3C10001). Antibody (PI-3000 (Vector l) in antibody diluent (DAKO, S0809) abs (Burlingame, CA) - 1 / 500) was applied to the slides for 1 hour, This was followed by chromogenic labeling with 3,3-diaminobenzidine (DAB) (DAKO, K3468). Slides were counterstained with hematoxylin, dehydrated, and stained with Vectamount (H- 5000, Vector Labs) to permanently fix the

[0163] Results: Kinetic analysis of monoclonal antibody BAMB31_2a (mIgG2a) revealed that A Affinity binding to the β3pE-40 peptide (SEQ ID NO: 40) was confirmed. Binding was detected when human Aβ-140 peptide (SEQ ID NO: 41) was applied at a concentration of 1 M. BAMB31_2 against human Aβ3pE-40 peptide (SEQ ID NO: 40) Sensorgrams (single cycle kinetics) showing the binding interaction of a(mIgG2a) are shown in Figure 1. The molecules used as the comparison standard were J&JPRD / Aβ / pE3 / 1 mIgG2a and mE8c mIgG2a (Figure 2) were evaluated in the same assay, and mE8c and J&JPRD The results demonstrated that BAMB31 has a higher affinity for Aβ / pE3 / 1 compared to Aβ / pE3 / 1. constant (affinity, K D ) and the kinetic constant (k a / k d ) are shown in Table 4.

[0164] [Table 5]

[0165] BAMB31_2a (mIgG2a) parent and BAMB246 (human IgG1 chimera) parent BAMB31 HFA with reduced risk of Hc CDR2 NG deamidation compared to Kinetic analysis of mAbs (BAMB674 and BAMB675) revealed that Aβ3pE-28 peptide The equilibrium binding constant (affinity) was shown to be sex, K D ) and the kinetic constant (k a / k d ) are shown in Table 5.

[0166] The aforementioned humanized 3pE-specific antibodies hE8L, R17L, R17, CI-C7, B12L , Antibody I, and Antibody II (U.S. Pat. No. 9,944,696 (B2) and U.S. Pat. No. 8,679,413). 498(B2) (previously discussed in BAMB3) as shown in Table 5. 1 The affinity of the HFA molecule is higher.

[0167] [Table 6] A human chimeric antibody has a mouse variable region on a human IgG1 constant region.

[0168] [Table 7] HU-3pE-β-amyloid concentration range: 0.6-4.5 nM

[0169] Higher affinity as measured by SPR also translated into improved plaque binding The primary antibodies were immunohistochemically stained on brain sections of transgenic mouse brains. A dilution series was performed. All antibodies showed visible proteins at 2 μg / mL, although in different ranges. Antibodies CI-C7 and R17L were used at a concentration of 0.1 μg / mL. In accordance with their low affinity as measured by SPR, they did not produce visible plaque labeling. At a concentration of 0.05 μg / mL, BAMB674 and BAMB675 inhibited the plaque markers. While showing a positive response, plaque labeling was not seen at this concentration relative to the control molecule (Figure 1). 3) At a concentration of 0.025 μg / mL, visible plaque labeling was observed for all molecules tested. (Almost completely) invisible.

[0170] In conclusion, the immunohistochemical data demonstrate that BAMB674 and We confirm the SPR data showing higher affinity for BAMB675 and BAMB675.

[0171] BAMB246 (human IgG1 chimera) and the current BAMB31 HFA mAb (B AMB674 and BAMB675) are highly homologous mouse Aβ 3pE-28 peptides. (SEQ ID NO: 46), as well as fibronectin, Aβ 1-28 Peptide (SEQ ID NO: 43), and Aβ 3pE-2 with scrambled amino acids 3-9 8 peptide (scrambled 3pE-28) (SEQ ID NO: 44) showed greater than 5 logs of selectivity. The results of selectivity and equilibrium binding constants (affinity) for related peptides and proteins were sex, K D ) and the kinetic constant (k a and k d ) are shown in Table 6.

[0172] [Table 8] Scrambled 3pE-28 is human Aβ 3pE with scrambled amino acids 3–9. -28 peptide. The selectivity fold was calculated by dividing the KD in Table 4 by the KD in Table 3. K ≧ 1.20E-06 D (M) Antigen binding was observed up to the maximum tested concentration of 1.2 μM. Not detected.

[0173] Wild-type IgG1 (BAMB674) and +YTE IgG1 isotype (BAMB6 75) are shown in Table 7. mAb (BAMB675) showed a pharmacokinetic profile similar to that of +YTE antibody in cynomolgus monkeys. Compared to wild-type IgG1 (BAMB674), which translates to a longer circulating half-life in humans and The affinity for cynomolgus monkey FcRn is approximately three times higher (Figure 11).

[0174] [Table 9] KD(M) values ​​were averaged from 5-6 independent replicates.

[0175] Example 6: Sandwich ELISA for cross-reactivity testing Selected Aβ3pE monoclonal antibody BAMB31 inhibits rodent Aβ3pE -40 (SEQ ID NO: 45) and human Aβ1-40 (SEQ ID NO: 41), Aβ1-42 (SEQ ID NO: No. 48), Aβ11pE-40 (SEQ ID NO: 49) and Aβ11pE-42 (SEQ ID NO: 50) ) was assessed using synthetic peptides. Using the combination of 40 / 28-HRPO, Aβ1-40 (SEQ ID NO: 41), Aβ1 Cross-reaction with 1pE-40 (SEQ ID NO: 49) and rodent Aβ3pE-40 (SEQ ID NO: 45) We investigated the reactivity of BAMB31+JRF / cAβ42 / 26-HRPO. The results were analyzed using the ELISA kit, which was used to detect the interaction between Aβ1-42 (SEQ ID NO: 48) and Aβ11pE-42 (SEQ ID NO: 50). Differential reactivity was investigated. Concentrations up to 10,000 pg / mL were tested.

[0176] Materials and Methods: Standards were prepared at 0.1 mg / mL in dimethyl sulfoxide (DMSO) (S The peptides were dissolved in IgG1 and stored at -80°C. The samples were further diluted to 1 pg / mL in 0.1% casein in BS. isorb ELISA plates (NUNC) were coated with 1.5 μg / mL of Coat overnight at 4°C with monoclonal antibody from BAMB31_1 at a concentration of 1 mL. The next day, the plates were washed and incubated with 0.1% casein in PBS for 1-4 hours at room temperature. The standard substance was HRPO-labeled secondary antibody (JRF / cAβ40 / 28-HR PO or JRF / cAβ42 / 26-HRPO) at 4°C overnight. After overnight incubation, wash the plates and incubate with TMB peroxide according to the manufacturer's recommendations. The assay was performed using a chlorinated EIA substrate kit (Biorad).

[0177] Results: BAMB31 inhibited Aβ3pE-40 (sequence 1) at concentrations up to 10 ng / mL. No. 40) and Aβ3pE-42 (SEQ ID NO: 51), and 40 (SEQ ID NO: 41), human Aβ1-42 (SEQ ID NO: 48) and rodent Aβ3pE-4 0 (SEQ ID NO: 45) and human AβpE11-40 (SEQ ID NO: 49) and AβpE11-4 2 (SEQ ID NO:50) (Figure 4).

[0178] Example 7: Antibody Reaction to Plaques in Transgenic Mouse and Human AD Brain Tissue Immunohistochemistry to test for reactivity Antibody reactivity to plaques was examined in formalin-fixed, paraffin-embedded (FFPE) brain tissue, This was investigated in both human and cryopreserved brain tissue.

[0179] Materials and Methods Formalin-fixed, paraffin-embedded brains: sections were deparaffinized for immunohistochemical analysis. After silencing and rehydration, the transgenic mouse brain was incubated in formic acid (70% in distilled water) for 10 min. Antigen retrieval was performed by incubating the slides to remove endogenous peroxidase activity. The staining was then bleached with 3% hydrogen peroxide (DAKO (Glostrup, Denmark) S2023). The sections were then treated with BAMB246 (huIgG1 chimera), BAMB674 or BAMB675 (working concentration: with background reduction component (DAKO, S3022) The cells were incubated with 4 μg / mL of antibody diluent for 1 hour. Afterwards, the secondary antibody labeled with HRP (PI in antibody diluent (DAKO, S0809)) was added. -3000, Vector labs-1 / 500) was applied to the slides for 1 hour, followed by Chromogenic labeling was performed with 3,3-diaminobenzidine (DAB) (DAKO, K3468). Slides were counterstained with hematoxylin, dehydrated, and stained with Vectamount (H-500 0, Vector Labs) to permanently fix the

[0180] Cryopreserved brain: Human brain samples were flash frozen and sliced ​​(20 μm thick) on a cryostat. The sections were stored at -80°C before use. They were then dried at room temperature and subsequently formalin-fixed and depleted of endogenous peptides. 3% hydrogen peroxide (DAKO (Glostrup, Denmark) S2 023), and PBS1x + 0.3% Triton X-100 and Blocked for 1 hour in 10% normal goat serum (DAKO, X0907). Antibody pE3 / 16 (antibody dilution with background reduction component (DAKO, S3022) A 2 μg / mL solution of HRP in diluent was applied to the sections for 1 h. After extensive washing, HRP was followed by conjugated anti-mouse secondary antibody (Envision, DAKO, K4000). The slides were then incubated with chromogenic DAB label (DAKO, K3468) for 1 h. Slides were counterstained with hematoxylin, dehydrated, and fixed in organic fixation medium (Vectamount The animals were fixed in a 3D microscope (Vector Labs). mer(Hamamatsu Photonics (Shizuoka, Japan) ) was held.

[0181] Results: BAMB264 (human IgG1 chimera) as well as BAMB674 and BAMB67 The reactivity of 5 was demonstrated on FFPE tissues from transgenic mice (Figures 5A to 5F). Furthermore, BAMB31_2a (mIgG2a) was expressed in the parenchyma of cryopreserved AD brain tissue. The results showed that the plaques detected by antibody 4G8 were consistent with those shown in Fig. 6. Fractions are also labeled by BAMB31 in cryosections of human brain (Figure 5A-C). 5F). ++

[0182] Example 8: Serum antibody titers after treatment in transgenic mice Serum antibody titers were investigated after treatment with BAMB31 and the control molecule mE8c.

[0183] Materials and Methods: Aged transgenic mice expressing high levels of human Aβ42 and Aβ40 peptides were cultured in vitro. Transgenic mice (22-23 months old) were immunized with 20 mg / kg of mE8c mIgG2a , BAMB31_2a (mIgG2a) or isotype control antibody mIgG2a (treatment group After antibody administration, mice were given a single intraperitoneal (ip) injection (n=5 per mouse). At 24 and 48 hours, the mice were placed in MICROVETTE® collection tubes ( 100Z and 300Z, Sarstedt (Numbrecht, Germany) At the time of sacrifice (4 days after injection), whole blood was collected via the great saphenous vein by orbital puncture. The collected whole blood was incubated at room temperature for 1-2 hours, followed by stirring at 10,000 rpm. Serum was separated from the blood clot by centrifugation for 10 min at 4° C. Serum antibody titers were determined by allotype Specific enzyme-linked immunosorbent assay (ELISA) For this purpose, Nunc MaxiSorp™ flat-bottom plates (T Hermo Scientific (Waltham, MA) at 1.5 μg / mL Mouse monoclonal anti-IgG2a(a) (BD Biosciences (San J mE8c mIgG2a, BAMB3 Blocking antibody standards 1_2a (mIgG2a) and isotype control mIgG2a 1 μg / mL in buffer (1% BSA in PBS buffer + 0.05% Tween-20) and further diluted to 0.1 ng / mL in blocking buffer. After washing (PBS Buffer + 0.05% Tween-20), and then block the samples with blocking buffer for 1 hour at room temperature. The standards and pre-diluted serum samples were then applied to the coated MAXISO Incubated in RP™ plate for 1 hour at room temperature. Sample incubation After washing the plate, Peroxidase-AffiniPure goat anti-mouse Ig G (Fcy Subclass 2a specific) antibody at room temperature for 1 hour. The plate was washed and the TMB peroxide EIA substrate kit (1 step, Pierce) was added. Color was developed by adding 2N H 2 SO 4 Adding The color development was stopped after 2 minutes and the plate was read on an EnVision multimode plate reader (Perk Plates were read at 450 nm using a 350 Hz read-only read (in Elmer).

[0184] Results: Serum antibody titers were measured 24, 48 and 64 hours after intraperitoneal injection of 20 mg / kg antibody. The mean serum antibody concentrations were measured at 24 hours and 4 days after administration for all antibodies examined. For BAMB31_2a and the isotype control antibody, the antibody concentrations were The decrease occurred gradually over time (down to about 30% at day 4), whereas at day 4, it was about 97% For the reduced mE8c, a clearly larger decrease could be observed over time. (Figure 7).

[0185] In conclusion, intraperitoneal administration of BAMB31_2a and mE8c in a mouse model of plaque deposition Differences in the pharmacokinetic profile following intraluminal injection were demonstrated, with BAMB31 compared to mE8c A slower clearance was noted after treatment with the _2a antibody.

[0186] Example 9: Long-term efficacy studies in transgenic mouse models In a transgenic mouse model, BAMB31_2a mIgG2a The efficacy of reducing amyloid burden after long-term treatment was investigated, as well as the effect on microbleeds.

[0187] Materials and Methods: PDAPP(V717F) transgenic mice (mean age at the start of the study The 18.3-month-old boy was treated with BAMB31_2a antibody (mIgG2a) at 30 mg / kg for 1 h. Treatment was by intraperitoneal injection once a week for 12 weeks. A control group was included in the experiment. The animals were euthanized 7 days after the last intraperitoneal injection (end of study). The mean age at the time of collection was 21.1 months. Mice were perfused with PBS prior to collection. The left hemisphere (fraction Fraction 1: hippocampus, fraction 2: remaining brain without hippocampus / cerebellum / brainstem) for further biochemical analysis. The right hemisphere was fixed overnight in a formalin-based fixative and then paraffin-free. The tissue was embedded in PET and sliced ​​(5 μm) using a microtome.

[0188] To assess the effect of long-term treatment on amyloid burden, biochemical and immunological analyses were performed. For biochemical analysis, Tallprep Lysin was used. g Using a Matrix D tube (MP Bio), ice-cold 5 M guanidine-HC1 l and in 50 mM Tris / HCl extraction buffer (100 mg tissue / mL extraction buffer). Brains were homogenized. After homogenization, samples were placed in an end-over-end rotating wheel for 3 h at room temperature. The resulting homogenates were then placed in - Stored at 80°C.

[0189] Synthetic Aβ peptide standards were prepared at 0.1 mg / mL in dimethyl sulfoxide (DMSO) ( Sigma) and stored at -80°C. Peptides were dissolved in 0.5 M GuHCl + 5 mM Tris-HCl, pH 8.0 (PBS) The GuHCl extract was further diluted with 10-fold dilution of extraction buffer in 0.1% casein. Freeze, dilute 1:10 in ice-cold 0.1% casein in PBS, and incubate at 4°C for 20 min at 20,000°C. The supernatant was collected for use in the sandwich MSD assay and centrifuged at 0 g. .5M GuHCl+5mM Tris-HCl-pH8.0 (0.1% casei in PBS) The samples were further diluted with 1:10 dilution of extraction buffer in PBS.

[0190] 96-well sector plate standard (Meso Scale Discovery(R (Mockville, MD) at a concentration of 1.5 μg / mL in PBS. The plates were coated with 0.1% cDNA in PBS overnight at 4°C. The next day, the plates were washed and Zein was used for blocking at room temperature for 2 hours. Standards and samples were then blocked with biotin-labeled secondary antibody. The plates were incubated with the antibody at 4°C overnight. After overnight incubation, the plates were washed. and a secondary detection reagent (Streptavidin-SULFO-TAG™ labeled). Both were incubated at room temperature for 2 hours. The plate was washed and washed with 2x Read Buffer. r T was added and the plate was then read according to the manufacturer's recommendations. 2 Weighted A four-parameter logistic model with a weighting function was used to estimate Aβ concentrations using the standard curve. The decision was made.

[0191] Brain homogenates using the JRF / AβN / 25+4G8 biotin antibody combination We investigated the Aβ1-x concentration in the blood.

[0192] For immunohistochemical analysis, sections were deparaffinized and rehydrated, then diluted with formic acid (7 mL in distilled water). Antigen retrieval was performed by incubating the slides in 0% CO for 10 min. Peroxidase activity was measured using 3% hydrogen peroxide (DAKO, Glostrup, Denmark). k, S2023). 2) in antibody diluent with biotinylated 4G8 antibody (Bio Sections were incubated overnight with legend (San Diego, Calif.). After washing the area, streptavidin-HRP (PK6100 Elite, Vector or labs) was applied to the slides for 30 min, followed by 3,3-diaminobenzidine (D Chromogenic labeling was performed with AB) (DAKO, K3468). Slides were counterstained with hematoxylin. The sections were stained, dehydrated, and reconstituted with Vectamount (H-5000, Vector Labs). The slides were permanently fixed in place using a NanoZoomer slide scanner (Hamamatsu Photonics). Images (20x) were generated using optics and analyzed using Matlab / Phaedra. The region of interest (ROI) was determined by the method described by Franklin and Pa xinos atlas(Franklin KB,Paxinos G.Mouse brain in stereotaxic coordinates.Waltham Each ROI was manually drawn according to the method described in (Academic Press, 1997). The percentage of DAB-labeled area per total area was calculated.

[0193] To assess the effect on microhemorrhage, Perl staining was performed. Paraffin-embedded tissue sections were then paraffinized with acid ferrocyanide solution according to the protocol described below. The ferric ion (Fe3+) present in the microbleed combines with ferrocyanide. Combined, these two compounds result in the formation of a blue pigment called Prussian blue. After rehydration, the cells were diluted with 2% potassium ferrocyanide (Sigma-Aldrich) and 2% ice Sections were incubated for 30 min in a 1 / 1 mixture of hydrochloric acid (Sigma-Aldrich). The slides were rinsed three times with distilled water and then stained with nuclear fast repellent. d (Sigma-Aldrich), followed by counterstaining with distilled water, dehydration, and fixation with ( The plates were then rinsed with Vectamount (Vectamount, Vector Labs). The scans were performed using a Zoomer slide scanner (Hamamatsu Photonics). The number of Perl positive cells near the meninges was counted manually.

[0194] Results: Overall, there were few clinically significant differences in baseline, isotype control, and BAMB31 treatment groups. Microbleeds were observed (Figure 8). Biochemical analysis showed that Aβ1-x concentrations in the hippocampus were , 34% (p<0.0001) of BAMB31_2a compared to the isotype control antibody In addition, immunohistochemistry using antibody 4G8 showed a decrease in the levels of IL-1 in the hippocampus and cortex. 23% (p<0.0001) and 37% (p The reduction was <0.001.

[0195] In conclusion, after chronic intraperitoneal injection of BAMB31 in a mouse model of plaque deposition The efficacy of the drug in reducing amyloid burden without causing an increase in the occurrence of microhemorrhages in This was demonstrated, showing a favorable efficacy to toxicity ratio following treatment with the BAMB31 antibody.

[0196] Example 10: Pharmacokinetics of BAMB31 HFA mAb +YTE IgG1 (BAMB675) isotype and wild-type IgG1 (BAMB6 74) The pharmacokinetics of BAMB31 HFA mAb was investigated in the peripheral circulation and brain. For direct comparison of sexes, evaluation was performed in cynomolgus monkeys.

[0197] Materials and Methods: Three animals per group were administered 25 mg / kg of each mAb intravenously (iv ) was administered by bolus injection and serum samples were collected over a 5-week period. Three additional monkeys were given an intravenous bolus injection of 25 mg / kg of Brain tissue was collected from each group on day 42. In total, brain tissue was collected on days 7 and 42 for each molecule. Brain samples were collected from three cynomolgus monkeys.

[0198] Drugs of BAMB674 and BAMB675 from in vivo cynomolgus monkey serum and brain tissue samples The exposure analysis was performed using Meso Scale Discovery (MSD) Sector I. Tailored electrochemiluminescence with endpoint determination on the mager S600 The assay was performed using the immunoassay (ECLIA) method. Each matrix was applied, resulting in four independent methods for measuring exposure. The format is as follows: mAb compounds are captured and anti-human Fc specific (CH2 domain) In) Detection was performed with mouse mAb. For brain tissue preparation, flash-frozen tissue was freeze-ground and buffered. Homogenates were made by dilution in broth. Protein concentrations of tissues were verified. The final protein concentration used in the method was determined using a BCA assay. Five-parameter logistic (auto-estimated) fitting with Y2 standard curve weighting Raw data regression was performed in Watson LIMS software using the ELISA kit.

[0199] Two compartments with intravenous (iv) administration (Admin) to the central compartment Mento (Center (V C ) and organization (V T ) compartmental) pharmacokinetic (PK) model, Using the intercompartmental clearance (Q) and linear clearance (CL), The PK of BAMB674 and BAMB675 is characterized in cinnamon-eating monkeys. A schematic diagram of the model is shown.

[0200] Results: The terminal half-life of each antibody was calculated and compared with the data from cynomolgus monkeys and the two compartments. The results are shown in Figure 11 along with the fitting of the ment model. BAMB675) is approximately 1. The half-life was increased by 6-fold compared to the wild-type IgG1 mAb (BAMB67 4) The FcRn affinity of the +YTE isotype (BAMB675) was increased by approximately 3-fold compared with that of the +YTE isotype (BAMB675). It is related.

[0201] Brain lysate levels across regions and between mAbs were similar at day 7. However, by day 42, only the YTE mAb was consistently detected across brain regions and animals. The results are shown in Figure 12, which indicates increased exposure of +YTE mAb at later time points. This is consistent.

[0202] In describing the present invention and various embodiments thereof, specific terminology is used for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected. It is not intended that the present invention be limited to the above-mentioned embodiments, and those skilled in the relevant art will be able to use other equivalent components. and that other methods may be developed without departing from the broad concept of the invention. All references cited throughout this specification are hereby incorporated by reference in their entirety. All such claims are incorporated by reference as if fully set forth herein.

Claims

1. a. SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; b. SEQ ID NOs: 1, 7, 3, 4, 5, and 6, respectively; c. SEQ ID NOs: 1, 7, 3, 8, 5, and 6, respectively; d. SEQ ID NOs: 1, 2, 3, 8, 5, and 6, respectively; e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively; f. SEQ ID NOs: 56, 57, 3, 4, 5, and 6, respectively; g. SEQ ID NOs: 56, 58, 3, 4, 5, and 6, respectively; h. SEQ ID NOs: 56, 7, 3, 8, 5, and 6, respectively; i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively; j. SEQ ID NOs: 56, 7, 3, 4, 5, and 6, respectively; k. SEQ ID NOs: 1, 57, 3, 4, 5, and 6, respectively; l. SEQ ID NOs: 1, 58, 3, 4, 5, and 6, respectively; or m. having the polypeptide sequences of SEQ ID NOs: 56, 2, 3, 4, 5, and 6, respectively , heavy complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 An isolated monoclonal antibody comprising (LCDR1), LCDR2, and LCDR3 or an antibody thereof The antibody or antigen-binding fragment thereof is 3pE Ab, preferably human An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to 3pE Ab.

2. SEQ ID NO: 9, 11, 13, 15, 16, 17, 19, 20, or 21 and at least A heavy chain variable region having a polypeptide sequence that is 95% identical to that of SEQ ID NO: 10, 12, 14, 18, 22, 53, or 55 having a polypeptide sequence at least 95% identical 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising a chain variable region.

3. a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:22 a light chain variable region having a peptide sequence; b. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:9 and a polypeptide of SEQ ID NO:10 a light chain variable region having a peptide sequence; c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:11 and a polypeptide sequence of SEQ ID NO:12 a light chain variable region having a peptide sequence; d. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:13 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; e. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:15 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; f. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:16 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; g. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:20 and a polypeptide sequence of SEQ ID NO:14 a light chain variable region having a peptide sequence; h. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:17 and the polypeptide sequence of SEQ ID NO:

18. a light chain variable region having a peptide sequence; i. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and a polypeptide sequence of SEQ ID NO:18 a light chain variable region having a peptide sequence; j. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and a polypeptide sequence of SEQ ID NO:53 a light chain variable region having a peptide sequence; k. A heavy chain variable region having the polypeptide sequence of SEQ ID NO:21 and the polypeptide sequence of SEQ ID NO:55 The isolated monoclonal antibody of claim 1, comprising a light chain variable region having a peptide sequence is an antigen-binding fragment thereof.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3 is chimeric. An isolated monoclonal antibody or an antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is human or humanized. An isolated monoclonal antibody or antigen-binding fragment thereof described in any one of claims 1 to 4.

6. a. a heavy chain amino acid sequence comprising SEQ ID NO:37 and a light chain amino acid sequence comprising SEQ ID NO:38 、 b. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:38 、 c. A heavy chain amino acid sequence comprising SEQ ID NO:37 and a light chain amino acid sequence comprising SEQ ID NO:52 or d. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:54 1. An isolated monoclonal antibody comprising:

7. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 is coated Isolated nucleic acid.

8. A vector comprising the isolated nucleic acid of claim 7.

9. A host cell comprising the vector of claim 8.

10. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a pharmaceutical and a pharma- ceutically acceptable carrier.

11. Inhibiting the formation of plaques containing β-amyloid protein in a subject in need thereof A method for treating a condition associated with the use of a compound according to any one of claims 1 to 6. or an antigen-binding fragment thereof, or a pharmaceutical composition according to claim 10. administering to said subject in need thereof.

12. 12. The method of claim 11, wherein the condition is Alzheimer's disease.

13. The condition is dementia associated with trisomy 21 (Down's syndrome), diffuse Lewy body syndrome, Hereditary cerebral amyloidosis with cerebrovascular disease, inclusion body myositis, cerebral amyloid angiopathy, and Dutch amyloidosis 12. The method of claim 11, wherein the patient is selected from the group consisting of: hemorrhage (HCHWA-D).

14. Methods for reducing plaques associated with Alzheimer's disease in a subject in need thereof - Patent application The method comprises the step of: administering to a subject in need thereof an antigen-binding fragment thereof, or a pharmaceutical composition according to claim 10. The method includes:

15. The present invention relates to a method for preventing the seeding activity of 3pE Aβ in a subject in need thereof. The method comprises using the monoclonal antibody or its derivatives according to any one of claims 1 to 6. Administering to a subject in need thereof an antigen-binding fragment thereof or a pharmaceutical composition according to claim 10. and

16. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

20. A method for producing a monoclonal antibody or antigen-binding fragment thereof comprising: Culturing cells containing nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof. and recovering the antibody or antigen-binding fragment thereof.

17. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. A method for producing a pharmaceutical composition, said method comprising administering to said monoclonal antibody or an antigen thereof combining the binding fragment with a pharma- ceutical acceptable carrier to obtain said pharmaceutical composition. method.

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