Antibodies against pyroglutamate amyloid-β and their use

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] This invention relates to an antibody against amyloid-beta (Aβ) peptide and a therapy using the antibody. Regarding the field of methods. Specifically, antibodies are used to identify and treat amyloid-related disorders. It can be used for this purpose.

[0002] (Reference to electronically submitted sequence listings) This application is filed under the name "JAB7013USPSP Sequence Listing" and dated March 11, 2019. The creation date is as follows, and it was submitted electronically via EFS-Web as an ASCII format sequence listing. Includes a sequence listing with a size of 76kb. Sequence listings submitted via EFS-Web are This is part of this specification, and the entirety is incorporated herein by reference. [Background technology]

[0003] Alzheimer's disease (AD) is a disease that gradually leads to a severe decline in mental function. Ultimately, this leads to death, affecting memory, cognition, logical thinking, judgment, and emotional stability. Alzheimer's disease is a degenerative brain disorder characterized by progressive loss of memory. It is a common cause of progressive mental disorders (dementia). Alzheimer's disease is confirmed worldwide. It is recognized and represents a major public health problem. This disease currently affects 5 million people in the United States alone. It is estimated that more than [number] individuals are affected. Currently, it is incurable, however... These treatments do not effectively prevent AD, nor do they reverse its symptoms or progression.

[0004] The brains of individuals with Alzheimer's disease show amyloid plaques and amyloid angiopathy (amyloid deposition in blood vessels). And they present with characteristic lesions called neurofibrillary tangles. The majority of these lesions, especially amylose Id plaques and neurofibrillary tangles are generally found in several areas of the brain that are important for memory and cognitive function. It can be seen. Amyloid plaques and amyloid angiopathy are also seen in trisomy 21 (Down syndrome). Hereditary intracerebral hemorrhage with diffuse Lewy body disease and Dutch-type amyloidosis (HCHW) Characterizes the brains of individuals with AD.

[0005] The main component of amyloid plaques is β-amyloid precursor protein (amyloid precur Various amyloid-beta proteins are produced by the cleavage of sor protein (APP). It is an Aβ peptide. The attachment of Aβ peptides in the brain is a disease that leads to AD. It is assumed to be an early and necessary step in the process, leading to changes in Aβ production. The amyloid precursor protein and presenilin gene cause familial early-onset Alzheimer's disease. Identifying mutations in this area suggests that changes in amyloid metabolism are central to the underlying pathogenic processes of this disease. This provides strong evidence that it is a typical phenomenon.

[0006] Amyloid-β peptide (3pE Aβ) having a pyroglutamate as the third residue is A 3pE Aβ is the main species that accumulates in the brains of patients with Alzheimer's disease. 3pE Aβ accounts for almost all diffuse cases in Alzheimer's disease. It is present in sexual plaques and mature plaques, is metabolically stable, and contributes to plaque seeding (pl It can play a role in both aque seeding and stabilization (Cynis et al.). ,Molecular Neurodegeneration,2016;11:48) Since no detectable levels of 3pE Aβ have been reported in CSF or plasma, target pepto This suggests that Tide is pathologically specific (DeMattos et al., Ne uron, 2012; 76: 1-13). Antibodies that selectively bind to 3pE Aβ may be useful for immunotherapy. It may be useful for immunotherapy.

Summary of the Invention

Means for Solving the Problems

[0007] As embodied and fully described, the present invention relates to antibodies and antigen-binding fragments thereof that bind to amyloid-β (3pE Aβ) having a pyroglutamate at the third residue, a method for generating an antibody or an antigen-binding fragment thereof that binds to 3pE Aβ, an assay method using such an antibody or an antigen-binding fragment thereof, and the use of an antibody or an antigen-binding fragment of the present invention for the manufacture of a medicament for treating, delaying the onset of, or restoring at least one pathological condition or symptom of Alzheimer's disease and other β-amyloid-related diseases. The antibodies of the present invention preferentially bind to Aβ peptides containing 3pE over Aβ peptides not containing 3pE. In certain embodiments, a. each of SEQ ID NO: 1, 2, 3, 4, 5, and 6, b. each of SEQ ID NO: 1, 7, 3, 4, 5, and 6, c. each of SEQ ID NO: 1, 7, 3, 8, 5, and 6, d. each of SEQ ID NO: 1, 2, 3, 8, 5, and 6, e. each of SEQ ID NO: 56, 57, 3, 8, 5, and 6, f. each of SEQ ID NO: 56, 57, 3, 4, 5, and 6,

[0008] In certain embodiments, a. each of SEQ ID NO: 1, 2, 3, 4, 5, and 6, b. each of SEQ ID NO: 1, 7, 3, 4, 5, and 6, c. each of SEQ ID NO: 1, 7, 3, 8, 5, and 6, d. each of SEQ ID NO: 1, 2, 3, 8, 5, and 6, e. each of SEQ ID NO: 56, 57, 3, 8, 5, and 6, f. each of SEQ ID NO: 56, 57, 3, 4, 5, and 6, g. each of SEQ ID NO: 56, 58, 3, 4, 5, and 6, h. each of SEQ ID NO: 56, 7, 3, 8, 5, and 6, i. each of SEQ ID NO: 1, 57, 3, 8, 5, and 6, j. each of SEQ ID NO: 56, 7, 3, 4, 5, and 6, k. Sequence numbers 1, 57, 3, 4, 5, and 6, respectively. l. Sequence IDs 1, 58, 3, 4, 5, and 6, respectively, or m. Each has the polypeptide sequences of sequence numbers 56, 2, 3, 4, 5, and 6, respectively. , heavy complementarity determining region 1 (HCDR1), H CDR2, HCDR3, Light chain complementarity determination region 1 Isolated monoclonal antibodies containing the ing region, LCDR1, LCDR2, and LCDR3 or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is 3pE Aβ, preferably This refers to an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to human 3pE Aβ. This is described herein.

[0009] In certain embodiments, the isolated monoclonal antibody or its antigen-binding fragment is SEQ ID NO: 9. 11, 13, 15, 16, 17, 19, 20, or 21 are at least 95% identical. Heavy chain variable region having a lipeptide sequence, or SEQ ID NOs: 10, 12, 14, 18, 22, 5 3. Includes a light chain variable region having a polypeptide sequence that is at least 95% identical to that of 55. nothing.

[0010] In certain embodiments, the isolated monoclonal antibody or its antigen-binding fragment is a. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly Light chain variable region having a peptide sequence, b. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and the polypeptide sequence of SEQ ID NO: 10 Light chain variable region having a ptide sequence, c. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11 and the poly Light chain variable region having a peptide sequence, d. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13 and the poly Light chain variable region having a peptide sequence, e. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and the poly Light chain variable region having a peptide sequence, f. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 16 and the poly Light chain variable region having a peptide sequence, g. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 20 and the poly Light chain variable region having a peptide sequence, h. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17 and the poly Light chain variable region having a peptide sequence, i. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19 and the poly chain of SEQ ID NO: 18 Light chain variable region having a peptide sequence, j. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly A variable region of a light chain having a peptide sequence, or k. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly It contains a light chain variable region having a peptide sequence.

[0011] In certain embodiments, the isolated monoclonal antibody or its antigen-binding fragment is a chimeric antibody. In certain embodiments, the isolated monoclonal antibody or its antigen-binding fragment is human or human. It has been converted to a typographical form.

[0012] In certain embodiments, the isolated monoclonal antibody is a. The heavy chain amino acid sequence of SEQ ID NO: 37 and the light chain amino acid sequence of SEQ ID NO: 38. b. Heavy chain amino acid sequence containing SEQ ID NO: 39 and light chain amino acid sequence containing SEQ ID NO: 38 , c. The heavy chain amino acid sequence of SEQ ID NO: 37 and the light chain amino acid sequence of SEQ ID NO: 52, or d. Heavy chain amino acid sequence including SEQ ID NO: 39 and light chain amino acid sequence including SEQ ID NO: 54 Includes.

[0013] In certain embodiments, the antigen-binding fragments are Fv, F(ab'), F(ab')2, and s A fragment consisting of cFv is selected from a group of fragments. The antibody or its antigen-binding fragment is 3pE Aβpe It selectively binds to ptide (e.g., Aβ3pE-40 and Aβ3pE-42) and other Aβ Cross-reactivity to peptides or β-amyloid precursor proteins (APP) is minimal. None, or not at all.

[0014] Isolation encoding a monoclonal antibody or its antigen-binding fragment disclosed herein. Nucleic acids are also provided.

[0015] The present invention comprises an isolated nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof. A cult is also provided.

[0016] The present invention comprises an isolated nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof. Host cells containing the actor are also provided. Furthermore, the isolated monoclonal antibody of the present invention or A hybridoma that generates antigen-binding fragments is also provided.

[0017] In certain embodiments, the isolated monoclonal antibody of the present invention or its antigen-binding fragment is used in conjunction with a pharmaceutical product. A pharmaceutical composition is provided, comprising a suitably acceptable carrier.

[0018] In the target population, the formation of plaques containing β-amyloid protein occurs. Methods for treating related conditions are also provided. The method involves the monoclonal antibody of the present invention Alternatively, administer the antigen-binding fragment or the pharmaceutical composition of the present invention to the target that requires it. Includes. In certain embodiments, the condition is Alzheimer's disease. The condition is dementia associated with trisomy 21 (Down syndrome) and diffuse Lewy body dementia. Inclusion body myositis, cerebral amyloid angiopathy, and hereditary encephalopathy with Dutch amyloidosis The group is selected from those consisting of blood (HCHWA-D).

[0019] A method for reducing plaques associated with Alzheimer's disease in subjects who require it. The method also provides the monoclonal antibody or its antigen-binding fragment, This includes administering the pharmaceutical composition of the present invention to a subject in need of it.

[0020] A method for preventing the seeding activity of 3pE Aβ in subjects where this is necessary is also available. The method is provided. The method involves the monoclonal antibody of the present invention or its antigen-binding fragment, or the present invention This includes administering the medicinal composition to a subject who requires it.

[0021] A method for producing a monoclonal antibody or an antigen-binding fragment thereof according to the present invention, the method is: Under conditions that produce a monoclonal antibody or its antigen-binding fragment, a monoclonal antibody or Culturing cells containing nucleic acids encoding the antigen-binding fragment, and the antibody or its antigen A method is also provided which includes recovering the bound fragments.

[0022] A method for producing the pharmaceutical composition of the present invention is also provided. The method involves the monoclona of the present invention. A pharmaceutical composition is obtained by combining an antibody or its antigen-binding fragment with a pharmaceutically acceptable carrier. This includes doing so.

[0023] One embodiment includes a kit and device comprising the above-mentioned antibody or its antigen-binding fragment.

[0024] Further objectives, features, and advantages of the present invention can be found in the detailed discussion of the following preferred embodiments. This will become clear to the contractors. [Brief explanation of the drawing]

[0025] [Figure 1] This is a sensorgram (single-cycle kinetics) of the affinity-binding interaction between BAMB31_2a (mIgG2a) and human Aβ(3pE-40) peptide, detected without surface plasmon resonance labeling. Gray trajectories represent double-reference subtraction data, while black trajectories represent fitted values. [Figure 2] This is a sensorgram (single-cycle kinetics) of the affinity-binding interaction between mE8c mIgG2a and human Aβ(3pE-40) peptide, detected without surface plasmon resonance labeling. Gray trajectories represent double-reference subtraction data, while black trajectories represent fitted values. [Figure 3A] This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparative reference, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3B] This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparison standard, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3C]This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparison standard, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3D] This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparison standard, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3E] This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparison standard, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3F] This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparison standard, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3G] This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparison standard, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3H] This figure shows the reactivity of BAMB674 and BAMB675, which serve as a comparison standard, to plaques as analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. [Figure 3I]This figure shows the reactivity of BAMB674 and BAMB675 against plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue, analyzed by immunohistochemistry. The results are shown for a primary antibody concentration of 0.05 μg / mL. Arrows indicate the plaque-labeled regions of 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] This graph shows the selectivity of BAMB31_1 when synthetic human Aβ peptides are detected in sandwich ELISA. [Figure 4B] This graph shows the selectivity of BAMB31_1 when synthetic human Aβ peptides are detected in sandwich ELISA. (A) Aβ1-40 (B) AβpE11-40. [Figure 5A] This figure shows the reactivity of BAMB246 (huIgG1 chimera) to plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue, as measured by immunohistochemistry. [Figure 5B] This figure shows the reactivity of BAMB246 (huIgG1 chimera) to plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue, as measured by immunohistochemistry. [Figure 5C] This figure shows the reactivity of BAMB674 to plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue, as measured by immunohistochemistry. [Figure 5D] This figure shows the reactivity of BAMB674 to plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue, as measured by immunohistochemistry. [Figure 5E]This figure shows the reactivity of BAMB675 to plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue, as measured by immunohistochemistry. [Figure 5F] This figure shows the reactivity of BAMB675 to plaques by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. Small insert panels show the entire stained brain section and enlarged areas. [Figure 6A] This figure shows the reactivity of cryopreserved AD brain tissue to plaques by immunohistochemistry using 4G8 at two different magnifications. [Figure 6B] This figure shows the reactivity of BAMB31_2a(mIgG2a) to plaques in cryopreserved AD brain tissue by immunohistochemistry at two different magnifications. [Figure 6C] This figure shows the reactivity of cryopreserved AD brain tissue to plaques by immunohistochemistry using 4G8 at two different magnifications. [Figure 6D] This figure shows the reactivity of BAMB31_2a(mIgG2a) to plaques in cryopreserved AD brain tissue by immunohistochemistry at two different magnifications. [Figure 7] This graph shows serum antibody concentrations at different time points after a single intraperitoneal (IP) administration of 20 mg / kg in transgenic mice. [Figure 8] This graph shows the number of Perl-positive cells and microbleeds in PDAPP mice, as evaluated by isotype control and after long-term treatment with BAMB31_2a(mIgG2a) antibody. [Figure 9] This graph shows the amyloid load after long-term treatment with BAMB31_2a(mIgG2a) antibody in the hippocampus of isotype control and PDAPP mice, as measured by an immunoassay detecting Aβ1X. Gray values ​​represent data points below the detection limit of the assay. [Figure 10] This is a schematic diagram of a two-compartment model for evaluating the PK characteristics of BAMB674 and BAMB675 monkeys. [Figure 11] This graph shows PK vs. observational data for BAMB674 and BAMB675. It displays blood concentrations of BAM31 HFA mAbs as +YTE IgG1 (YTE serum) isotype and wild-type IgG1 (WT serum) after intravenous (IV) bolus administration of 25 mg / kg in cynomolgus monkeys. The anti-Aβ 3pE antibody (3pE-AB) μg / mL concentration is shown over time on a logarithmic scale on the Y-axis, with days on the X-axis. The calculated half-life (t1 / 2) for each mAb is shown in the inserted text. [Figure 12] This graph shows the observed brain concentrations of BAMB674 and BAMB675. It also shows brain lysate levels of BAMB31 HFA mAb as +YTE IgG1 (YTE brain) isotype and wild-type IgG1 (WT brain) at days 7 and 42 after a 25 mg / kg IV bolus administration in cynomolgus monkeys. The anti-Aβ 3pE antibody (3pE-AB) μg / mL concentration is shown over time on a logarithmic scale on the Y-axis, with days on the X-axis. [Modes for carrying out the invention]

[0026] In the background technology and throughout this specification, various publications, articles, and patents are cited or The full contents of each of these references are incorporated herein by reference. The discussion of documents, operations, materials, devices, articles, etc. included in the details is within the context of the present invention. This is to give an answer. Such considerations are based on the fact that any or all of these things are disclosed or This allows it to constitute part of the prior art to any of the claimed inventions. isn't it.

[0027] The present invention is not limited to specific methods, reagents, compounds, compositions, or biological systems. Rather, it should be understood that these are changeable. Also, the terms used herein The terms used are for the sole purpose of describing specific embodiments and are not intended to be restrictive in any way. Please also understand that this is not the case.

[0028] Unless otherwise specified, all technical and scientific terms used herein are defined in accordance with the present invention. It has the same meaning as generally understood by those skilled in the art to which it belongs. Otherwise, Certain terms used herein have the meanings set forth herein.

[0029] When used herein and in the appended claims, the singular forms "a", "an", and " It is important to note that "the" can refer to multiple objects unless it is particularly clear from the context. There is.

[0030] Unless otherwise stated, the term “at least” preceding a set of elements means all of the set. It should be understood that this refers to the elements of the subject. Those skilled in the art will know that using only ordinary experimental procedures... By doing so, one may recognize many equivalents to the specific embodiments described herein, or This can be confirmed. Such equivalents are intended to be included by the present invention. It can be done.

[0031] When used in this specification, the terms "comprises" and "comprising" are used interchangeably. "includes", "include", "has", "having )", "contains", or "contains", or any of these Other variations of this intention are intended to include the integers or groups of integers mentioned, but these It does not exclude any other integers or groups of integers, and is non-exclusive or non-restrictive. The intention will be understood. For example, a composition, mixture, process, which includes a series of elements. Methods, articles, or apparatus are not necessarily limited to those elements only, but expressly... Any composition, mixture, process, method, article, or apparatus not listed herein, or which is not included in this list, is included in this list. It may include other elements that have not been present. Furthermore, unless explicitly stated otherwise, "or" " or " refers to a comprehensive "or" and not an exclusive "or". For example Condition A or B is such that A is true (or exists) and B is false (or does not exist). In the case where A is false (or does not exist) and B is true (or exists), and If both A and B are true (or exist), then the condition is satisfied by either one of them.

[0032] As used herein, the connecting term "and / or" between multiple enumerated elements means It is understood to encompass both individual and combined options. For example, two When the elements are connected by "and / or", the first option is the second option without the second element. This indicates that element 1 is applicable. The second option is that element 2 is applicable without element 1. This refers to applicability. The third option is that the first and second elements are applicable together. This refers to the act of doing so. One of these options is included in the meaning, and therefore, this When used in a specification, it is understood that the requirements of the term "and / or" are met. Options The simultaneous applicability of two or more of these is also included in the meaning, and therefore the term "and / or" It is understood that the requirements are met.

[0033] When used herein, the term "consists of" or "consist" Variations such as "of)" or "consisting of" are used in the specification and claims. When used across the entire range, it includes any enumerated integer or set of integers, but additional This indicates that integers or groups of integers will not be added to the specified method, structure, or composition.

[0034] When used herein, the term "consists essentially of" or This means "to essentially become of" or "to essentially become of" Variations such as "isting essentially of" are used throughout the specification and claims. When used, it includes any enumerated integer or group of integers, and optionally specifies a method and structure. , or any enumerated integer or that does not substantially alter the basic or novel properties of the composition This demonstrates that integer groups are also included. See MPEP §2111.03.

[0035] antibody The present invention is particularly preferred over 3pE-containing Aβ peptides. The present invention provides an antibody or its antigen-binding fragment that binds to 3pE Aβ peptide. or a method for generating the antigen-binding fragment thereof, and an antibody or so that binds to the 3pE Aβ peptide The present invention further provides a method for generating hybridomas that produce antigen-binding fragments. Methods for treating Alzheimer's disease and other β-amyloid-related diseases in individuals, A method for removing plaque associated with Zheimer's disease or other β-amyloid-related diseases, and The present invention includes methods for inhibiting the plaque-seeding activity of 3pE Aβ. The present invention also includes the following: We provide kits and devices containing antibodies or antigen-binding fragments thereof for use in a manner that is described. ru.

[0036] According to a particular embodiment, the present invention is a. Sequence numbers 1, 2, 3, 4, 5, and 6, respectively b. Sequence numbers 1, 7, 3, 4, 5, and 6, respectively c. Sequence numbers 1, 7, 3, 8, 5, and 6, respectively d. Sequence numbers 1, 2, 3, 8, 5, and 6, respectively e. Sequence numbers 56, 57, 3, 8, 5, and 6, respectively. f. Sequence numbers 56, 57, 3, 4, 5, and 6, respectively. g. Sequence IDs 56, 58, 3, 4, 5, and 6, respectively h. Sequence numbers 56, 7, 3, 8, 5, and 6, respectively. i. Sequence numbers 1, 57, 3, 8, 5, and 6, respectively j. Sequence numbers 56, 7, 3, 4, 5, and 6, respectively. k. Sequence numbers 1, 57, 3, 4, 5, and 6, respectively. l. Sequence IDs 1, 58, 3, 4, 5, and 6, respectively, or m. Each has the polypeptide sequences of sequence numbers 56, 2, 3, 4, 5, and 6, respectively. , Multicomplementarity Determination Region 1 (HCDR1), HCDR2, HCDR3, Light Chain Complementarity Determination Region 1 (LCDR1), LCDR2, and LCDR3 isolated monoclonal antibodies or their anti- The protoconjugated fragment is an antibody or its antigen-binding fragment, preferably 3pE Aβ, and more preferably human 3p This relates to an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to E Aβ.

[0037] According to another specific embodiment, the present invention relates to sequence numbers 9, 11, 13, 15, 16, 17, 1 Heavy chain variable regions having polypeptide sequences that are at least 95% identical to 9, 20, or 21 The region, or sequence numbers 10, 12, 14, 18, 22, 53, or 55 and at least 95 % Isolated monoclonal antibody containing a light chain variable region having the same polypeptide sequence or Regarding antigen-binding fragments.

[0038] According to another specific embodiment, the present invention is l. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly Light chain variable region having a peptide sequence, m. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and the polypeptide sequence of SEQ ID NO: 10 Light chain variable region having a ptide sequence, n. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11 and the poly Light chain variable region having a peptide sequence, o. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13 and the poly Light chain variable region having a peptide sequence, p. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and the poly 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 poly Light chain variable region having a peptide sequence, r. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 20 and the polynucleotide sequence of SEQ ID NO: 14 Light chain variable region having a peptide sequence, s. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17 and the poly Light chain variable region having a peptide sequence, t. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19 and the poly Light chain variable region having a peptide sequence, u. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the polynucleotide sequence of SEQ ID NO: 53 A variable region of a light chain having a peptide sequence, or v. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly Isolated monoclonal antibodies or antigen-binding fragments thereof containing a light chain variable region having a peptide sequence. Regarding.

[0039] In one embodiment, the present invention relates to Sequence IDs 1, 2, 3, 4, 5, and 6, respectively, or the same. These are sequence numbers 56, 58, 3, 4, 5, and 6, respectively, or sequence numbers 56, 2, 3, and 4, respectively. , 5, and 6, or polypeptide sequences of sequence numbers 1, 58, 3, 4, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, which includes a single The monoclonal antibody or its antigen-binding fragment is preferably at least 85% of SEQ ID NO: 21. Or 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or This is a heavy chain variable region having 99% identical polypeptide sequences, and SEQ ID NOs. 22 or 53 or 55 and at least 85%, preferably 90%, more preferably 95% or more, for example 95% , light chain variable regions having 96%, 97%, 98%, or 99% identical polypeptide sequences Includes. Preferably, the isolated monoclonal antibody or its antigen-binding fragment is the same as in SEQ ID NO: 21. A heavy chain variable region having a lipeptide sequence and polypeptide of SEQ ID NO: 22, 53, or 55 It includes a light chain variable region having an array.

[0040] In one embodiment, the present invention relates to Sequence IDs 1, 2, 3, 4, 5, and 6, respectively, or the same. These are sequence numbers 56, 58, 3, 4, 5, and 6, respectively, or sequence numbers 56, 2, 3, and 4, respectively. , 5, and 6, or polypeptide sequences of sequence numbers 1, 58, 3, 4, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, which includes a single The monoclonal antibody or its antigen-binding fragment is preferably at least 85% of SEQ ID NO: 20. Or 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or This has a heavy chain variable region with 99% identical polypeptide sequences, and at least one of the sequences of SEQ ID NO: 14. 85%, preferably 90%, more preferably 95% or higher, for example 95%, 96%, 97% It contains a light chain variable region having 98% or 99% identical polypeptide sequences. Preferably The isolated monoclonal antibody or its antigen-binding fragment contains the polypeptide sequence of SEQ ID NO: 20 It includes a heavy chain variable region and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14.

[0041] In one embodiment, the present invention relates to Sequence IDs 1, 7, 3, 4, 5, and 6, respectively, or the same. These are sequence numbers 56, 57, 3, 4, 5, and 6, respectively, or sequence numbers 56, 7, 3, and 4. , 5, and 6, or polypeptide sequences of sequence numbers 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, comprising 3. In another embodiment, The isolated monoclonal antibody or its antigen-binding fragment is at least 85% of SEQ ID NO: 19. Ideally 90%, more preferably 95% or higher, for example 95%, 96%, 97%, 98%. Or a heavy chain variable region having 99% identical polypeptide sequences, and at least SEQ ID NO: 18 Also 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% It contains a light chain variable region having %, 98%, or 99% identical polypeptide sequences. Preferably The isolated monoclonal antibody or its antigen-binding fragment is the polypeptide sequence of SEQ ID NO: 19. Includes a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18. .

[0042] In one embodiment, the present invention relates to Sequence IDs 1, 7, 3, 4, 5, and 6, respectively, or the same. These are sequence numbers 56, 57, 3, 4, 5, and 6, respectively, or sequence numbers 56, 7, 3, and 4. , 5, and 6, or polypeptide sequences of sequence numbers 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, comprising 3. In another embodiment, The isolated monoclonal antibody or its antigen-binding fragment is at least 85% of SEQ ID NO: 17. Ideally 90%, more preferably 95% or higher, for example 95%, 96%, 97%, 98%. Or a heavy chain variable region having 99% identical polypeptide sequences, and at least SEQ ID NO: 18 Also 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% It contains a light chain variable region having %, 98%, or 99% identical polypeptide sequences. Preferably The isolated monoclonal antibody or its antigen-binding fragment is the polypeptide sequence of SEQ ID NO: 17. Includes a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18. .

[0043] In one embodiment, the present invention relates to Sequence IDs 1, 7, 3, 4, 5, and 6, respectively, or the same. These are sequence numbers 56, 57, 3, 4, 5, and 6, respectively, or sequence numbers 56, 7, 3, and 4. , 5, and 6, or polypeptide sequences of sequence numbers 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, which includes a single The monoclonal antibody or its antigen-binding fragment is preferably at least 85% of SEQ ID NO: 16. Or 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or This has a heavy chain variable region with 99% identical polypeptide sequences, and at least one of the sequences of SEQ ID NO: 14. 85%, preferably 90%, more preferably 95% or higher, for example 95%, 96%, 97% It contains a light chain variable region having 98% or 99% identical polypeptide sequences. Preferably The isolated monoclonal antibody or its antigen-binding fragment contains the polypeptide sequence of SEQ ID NO: 16. It includes a heavy chain variable region and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14.

[0044] In one embodiment, the present invention relates to Sequence IDs 1, 7, 3, 4, 5, and 6, respectively, or the same. These are sequence numbers 56, 57, 3, 4, 5, and 6, respectively, or sequence numbers 56, 7, 3, and 4. , 5, and 6, or polypeptide sequences of sequence numbers 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, comprising 3. In another embodiment, The isolated monoclonal antibody or its antigen-binding fragment is at least 85% of SEQ ID NO: 15. Ideally 90%, more preferably 95% or higher, for example 95%, 96%, 97%, 98%. Or a heavy chain variable region having 99% identical polypeptide sequences, and at least SEQ ID NO: 14 Also 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% It contains a light chain variable region having %, 98%, or 99% identical polypeptide sequences. Preferably The isolated monoclonal antibody or its antigen-binding fragment is the polypeptide sequence of SEQ ID NO: 15. Includes a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 14 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14. .

[0045] In one embodiment, the present invention relates to Sequence IDs 1, 7, 3, 4, 5, and 6, respectively, or the same. These are sequence numbers 56, 57, 3, 4, 5, and 6, respectively, or sequence numbers 56, 7, 3, and 4. , 5, and 6, or polypeptide sequences of sequence numbers 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, comprising 3. In another embodiment, The isolated monoclonal antibody or its antigen-binding fragment is at least 85% of SEQ ID NO: 13. Ideally 90%, more preferably 95% or higher, for example 95%, 96%, 97%, 98%. Or a heavy chain variable region having 99% identical polypeptide sequences, and at least SEQ ID NO: 14 Also 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% It contains a light chain variable region having %, 98%, or 99% identical polypeptide sequences. Preferably The isolated monoclonal antibody or its antigen-binding fragment is the polypeptide sequence of SEQ ID NO: 13. Includes a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 14 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14. .

[0046] In one embodiment, the present invention relates to Sequence IDs 1, 7, 3, 8, 5, and 6, respectively, or thereafter. These are sequence numbers 56, 57, 3, 8, 5, and 6, respectively, or sequence numbers 56, 7, 3, and 8, respectively. , 5, and 6, or polypeptide sequences of sequence numbers 1, 57, 3, 8, 5, and 6, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR The present invention relates to an isolated monoclonal antibody or its antigen-binding fragment, comprising 3. In another embodiment, The isolated monoclonal antibody or its antigen-binding fragment is at least 85% of SEQ ID NO: 11. Ideally 90%, more preferably 95% or higher, for example 95%, 96%, 97%, 98%. Or a heavy chain variable region having 99% identical polypeptide sequences, and at least SEQ ID NO: 12 Also 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97% It contains a light chain variable region having %, 98%, or 99% identical polypeptide sequences. Preferably The isolated monoclonal antibody or its antigen-binding fragment is the polypeptide sequence of SEQ ID NO: 11. Includes a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 12 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12. .

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

[0048] In another specific embodiment, the isolated monoclonal antibody is a. Heavy chain amino acid sequence including SEQ ID NO: 37 and light chain amino acid sequence including SEQ ID NO: 38 , b. Heavy chain amino acid sequence containing SEQ ID NO: 39 and light chain amino acid sequence containing SEQ ID NO: 38 , c. Heavy chain amino acid sequence including SEQ ID NO: 37 and light chain amino acid sequence including SEQ ID NO: 52 , or d. Heavy chain amino acid sequence including SEQ ID NO: 39 and light chain amino acid sequence including SEQ ID NO: 55 Includes.

[0049] According to another specific embodiment, the present invention relates to the isolated monoclonal antibody or its antigenic antibody of the present invention. Regarding the combined fragment, the antibody or its antigen-binding fragment is a chimeric entity.

[0050] According to another specific embodiment, the present invention relates to the isolated monoclonal antibody or its antigenic antibody of the present invention. With respect to the combined fragment, the antibody or its antigen-binding fragment is human or humanized.

[0051] According to another specific embodiment, the present invention relates to an antigen-binding fragment, wherein the antigen-binding fragment is Fv, F( Selected from the group consisting of ab'), F(ab')2, and scFv. Antibody or so The antigen-binding fragment is a 3pE Aβ peptide (e.g., Aβ3pE-40 and Aβ3pE- 42) selectively binds to other Aβ peptides or β-amyloid precursor proteins (AP There is little to no cross-reactivity with P).

[0052] In another general aspect, the present invention relates to the monoclonal antibody or its antigen-binding fragment. Regarding isolated nucleic acids that encode [something]. Without altering the amino acid sequence of the protein, It is possible to change the code sequence of the protein (for example, by replacing, deleting, or inserting). Those skilled in the art will understand that this is possible. Therefore, by changing the amino acid sequence of a protein Without any modification, the nucleic acid encoding the monoclonal antibody or its antigen-binding fragment of the present invention Those skilled in the art will understand that the array can be modified.

[0053] In another general aspect, the present invention relates to the monoclonal antibody or its antigen-binding fragment. Regarding vectors containing isolated nucleic acids encoding plasmids, cosmids, and phages. Any vector known to those skilled in the art from the perspective of this disclosure, such as a vector or viral vector. - can also be used. In some embodiments, the vector is a set such as a plasmid. It is an alternative expression vector. The vector consists of, for example, a promoter and a ribosome-binding element. Conventional expression vectors include terminators, enhancers, selection markers, and origins of replication. It may include any elements to establish its function. The promoter is always express. It may be a type, inducible, or remodelable promoter. It can deliver nucleic acids to cells. Numerous expression vectors are known in this technology that allow antibodies or their antigens to bind within cells. Conventional cloning techniques can be used herein to generate composite fragments. Alternatively, a recombinant expression vector according to the embodiments of the present invention may be generated using artificial gene synthesis. It is possible.

[0054] In another general embodiment, the present invention relates to the monoclonal antibody or its antigen binding. This relates to a host cell containing an isolated nucleic acid encoding a fragment. As described herein, it is known to those skilled in the art. Any host cell present in this state can be used for recombinant expression of the antibody or its antigen-binding fragment of the present invention. This is possible. In some embodiments, the host cell is E. coli TG1 or BL21 cell. (For example, in the case of scFv or Fab antibody expression), CHO-DG44 or CHO- These are K1 cells or HEK293 cells (for example, in the case of full-length IgG antibody expression). According to a specific embodiment, the recombinant expression vector is designed to allow recombinant nucleic acids to be effectively expressed. Stable integration into the principal cell genome, chemical transfection, heat shock, or The host cells are transformed by conventional methods such as electroporation.

[0055] In another general aspect, the present invention relates to the monoclonal antibody or its antigen-binding fragment. A method for generating the monoclonal antibody or its antigen-binding fragment according to the present invention. Under these conditions, a fragment containing the nucleic acid encoding the monoclonal antibody or its antigen-binding fragment. Culturing cells and obtaining the antibody or the The present invention relates to a method comprising recovering antigen-binding fragments, expressed antibodies or their antigen-binding Fragments are collected from cells and, according to the prior art known in the art, It can be refined as described in the detailed document.

[0056] The present invention provides an isolated antibody or its antigen-binding fragment that binds to 3pE Aβ. In this specification, the term "antibody" means an immunoglobulin capable of binding to an antigen or a portion thereof. Immunoglobulins that can specifically bind to brin proteins, particularly 3pE Aβ. It refers to protein. The binding of antibodies to antigens can be measured by methods known to those skilled in the art. One example is the use of BIAcore™ equipment. Generally speaking, antibodies or The antigen-binding antibody fragment has a dissociation constant of 1 μM or less, preferably 100 nM or less, most preferably It is said that when the concentration is 10 nM or less, it specifically binds to the antigen.

[0057] The antigen-binding fragment of an antibody can bind to the antigen to which the intact antibody binds, thus enabling antigen binding. This refers to an antibody fragment that competes with the intact antibody. The antigen-binding fragment enables antigen binding. It contains a portion of the intact antibody (i.e., the variable region of the intact antibody) as an antigen-binding fragment. This is not limited to Fab, Fab', F(ab')2, Fv fragment, disulf Disulfide-stabilized Fv fragment (dsFv), (dsFv)2, dual fragment Heterozygous dsFv (dsFv-dsFv'), single-chain antibody molecule (e.g., scFV), diabole Di, minibody, nanobody, linear antibody, single-domain antibody (SDAB), camelized single-domain antibody, multispecific antibody formed from antibody fragments This includes any other antibody fragments that bind to the body and antigens but do not contain a complete antibody structure. can.

[0058] An antibody consists of two heavy chains and two light chains. Each heavy chain contains one variable domain or region. (V H ), followed by a steady domain or region (C H 1) Hinge region, and two further steady 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 ) has variable doping of heavy and light chains. The index or region contains antibodies (similarly similar to tablets) that are specific to a particular epitope (a structure similar to a key). It forms a paratope (structure) and precisely binds the paratope and epitope together. This makes it possible. Within the variable domain, there are three variable loops in the light chain and heavy chain, β -The strands are responsible for binding to the antigen. These loops are the complementarity-determining regions (CDRs). These are referred to as CDR1, CDR2, and CDR3.

[0059] CDRs are defined as complementarity-determining regions of antibodies. These are primarily involved in binding to antigens. These are the hypervariable regions of the heavy and light chains of the antibody. There are three CDRs (CDR1, CDR2, and CDR3). Light chain variable complementarity determination region The regions are also referred to as LCDR1, LCDR2, and LCDR3, and are heavy-chain variable complementary determinations. The specified regions are also referred to as HCDR1, HCDR2, and HCDR3. Antibody CDR CDR can be defined in many ways. For example, CDR in the variable region is defined in Kabat, Chot hia, definition of IMGT, and / or definition of three-dimensional structure, or C known in the art It can be identified according to any method of DR determination. The antibody CDR is Kabat (Kabat et al., 1992, Sequences of Proteins of Imm unological interest, 5th ed.,Public Healt Originally defined by the Service of the United Nations (NIH, Washington DC) The hypervariable region, Chothia (Chothia et al., Nature 34 The structural loop structure originally described by 2:877-883 (1989), or the independent 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 It can be identified as 22(2015).

[0060] When used in the context of antibodies, "isolated" means "by human hands" or any other method. This means that it has changed from its natural state, i.e., if isolation occurred naturally, then its original state It is changed from the environment, removed, or both. For example, Naturally occurring antibodies present in animals living in their natural state have "not been isolated," however The same antibodies isolated from existing materials in their natural state are "isolated." When the term is used, for example, "isolated antibody" refers to an antibody that has different antigen specificity. Antibodies that substantially do not contain antibodies (i.e., isolated antibodies that specifically bind to 3pE Aβ) The term "antibody" can refer to antibodies that do not bind to 3pE Aβ (substantially excluding antibodies that do not bind to 3pE Aβ). This can occur in compositions that do not spontaneously occur, such as immunoassay reagents, and this specification When this term is used in writing, within the scope of its meaning, it refers to the isolation of a composition. It can remain in the state of being an antibody.

[0061] A method for producing antibodies involves inoculating a host with a desired immunogen. A suitable host is... This includes mice, rats, hamsters, guinea pigs, rabbits, chickens, donkeys, horses, and monkeys. Chimpanzees, orangutans, gorillas, humans, and those capable of eliciting a mature immune response Any species that can be immunized can be mentioned, but are not limited to these. The immunization procedure is relevant to the art. It is established as "The Immunoassay Handbook", 2nd Edition, edited by David Wild(Nature Pub This has been described in numerous papers and publications, including (Lyricing Group, 2000). ru.

[0062] Preferably, the immunogen embodying the features of the present invention is a host target, for example, an animal or a human. It is administered in combination with an adjuvant. Suitable adjuvants include Freund's ( Freund's adjuvant, powdered aluminum hydroxide (alum), combined with Bordetella pertussis. Aluminum hydroxide and monophosphoryl lipid A-synthetic trehalose dicholinomicole Examples include, but are not limited to, T.

[0063] Typically, an immunogen or a combination of immunogen and adjuvant is administered subcutaneously to one or more cells. Alternatively, it is injected into a mammalian host by intraperitoneal injection. Preferably, an immunization program The procedure is carried out for at least one week, more preferably for two weeks or more. Polyclonal antibodies produced in this manner utilize methods well known in the art. It can then be isolated and purified.

[0064] Monoclonal antibodies are described in Kohler and Milstein's *Nature*, for example. Produced by the established hybridoma method described in 256:495~497 (1975). Yes, it is possible. Hybridoma methods typically involve immunizing host or host-derived lymphocytes. To do so, and to secrete lymphocytes, or monoclonal antibodies that have the potential to secrete them. The process involves harvesting, fusing lymphocytes with immortalized cells, and creating the desired monoclonal antibody This involves selecting cells that secrete bodily fluids.

[0065] The host is immunized and produces antibodies specific to the immunogen, or does not produce antibodies. It is possible to induce lymphocytes that can do this. Alternatively, lymphocytes can be immunized in vitro. It is also possible to use peripheral blood lymphocytes if human cells are desired. Splenocytes or lymphocytes derived 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, such as polyethylene glycol. Yes, there are. Examples include mutant rodents, cattle, or human myeloma that have been immortalized through transformation. Cells can be used. In contrast to non-fusion immortalized cells, hybridoma cells are parenchyma. A pure population is preferred. Therefore, after fusion, for example, hypoxanthine guanine phosphate By using mutant myeloma cells lacking boribosyltransferase (HGPRT), non-fusion can be achieved. Cells can be grown in a suitable culture medium that inhibits the proliferation or survival of immortalized cells. In cases like the one described, hypoxanthine, aminopterin, and thymidine are added to the culture medium (HAT When added to the culture medium, it inhibits the proliferation of HGPRT-deficient cells and simultaneously inhibits the proliferation of hybridomas. It can enable proliferation.

[0067] Preferably, immortalized cells fuse efficiently and are mixed together by selecting a culture medium such as HAT. It can be isolated from the group and supports stable, high levels of antibody expression after fusion. Preferred immortalization. The cell line is from the American Type Culture Collection, Ma Includes myeloma cell lines available from nassas,VA.

[0068] One aspect of the present invention is capable of producing monoclonal antibodies that bind to amyloid-beta peptides. This is a method for generating hybridoma cell lines. Such methods are generally known to those skilled in the art. Generally, this involves (i) selecting a host for antibody production and (ii) providing the host with the desired (iii) Inoculating with an immunogen and producing monoclonal antibodies that bind to the immunogen To create fusion cells that can do this, the inoculated host-derived cell line is subjected to continuous division. (iv) fusing the cells with (iv) cloning the fused cells to form a hybridoma cell line This includes obtaining and

[0069] One method of the present invention can produce a monoclonal antibody that binds to 3pE Aβ peptide. This includes generating hybridoma cell lines. The hybridomas are Freund Adjuvant cells. The first intraperitoneal injection of a desired immunogen, such as an Aβ peptide containing pyroglutamate, Next, for example, with additional immunization injections every 1-2 weeks, hybrid mice such as Balb / c mice are... It can be produced by immunizing animals capable of producing the cytoplasm. Subsequent isolation The fusion of the spleen is preferably performed using any technique commonly known to those skilled in the art, Kohler and Milstein modification procedures (Eur.J.Immunol.,197) This can be performed using SP2 / 0 cells as described in 6;6:292-295. Screening for a hybrid that produces antibodies specific to 3pE Aβ peptide It is possible to determine the cause. Screening is performed using a standard assay such as ELISA or RIA assay. This can be performed using a standard assay. One aspect of the present invention involves the monoclonal antibody BAMB3 A method for generating hybridoma cell lines that produce 1_1 or its humanized version. .

[0070] Monoclonal antibodies are described, for example, in U.S. Patent No. 4,166,452. It can also be produced by genetic engineering methods known in the relevant field. Monoclonal antibodies are produced using conventional procedures, for example, heavy and light antibody chains from mice. A specific antibody against Aβ having pyroglutamates, which specifically binds to the gene. We will search for DNA isolated from monoclonal antibody hybridoma cell lines that secrete antibodies. They can be isolated and sequenced using oligonucleotide explorers.

[0071] Antibody fragments containing a specific binding site for amyloid-beta peptide can also be generated. Such fragments can be produced by pepsin digestion of antibody molecules, such as F(ab' This can be generated by reducing the disulfide bridges of the )2 fragment and the F(ab')2 fragment. Examples of Fab fragments include, but are not limited to, those having the desired specificity. To enable rapid and easy identification of monoclonal Fab fragments, Fab expression It may be possible to construct an Ibrali (Huse et al., Science 256:1270) -1281(1989)). Fab, Fv, and ScFv antibody fragments all have odors associated with E. coli. It is expressed and can be secreted by E. coli, enabling the mass production of these fragments. Alternatively, F The ab'-SH fragment was directly recovered from E. coli, chemically linked, and then F(ab')2 fragment. It can form fragments (Carter et al., BioTechnology) 10:163-167 (1992). Other techniques for generating antibody fragments are known to those skilled in the art. It is knowledge. Single-chain Fv fragments (scFv) are also conceivable (e.g., US 5,761 (See Nos. 894 and 5,587,458). The Fv and sFv fragments have a steady-state region. It is the only species that possesses a complete combination site that does not involve nonspecific binding. A decrease is highly likely to be shown. For example, antibody fragments are, for example, U.S. 5,642,87 It may also be a "linear antibody" as described in Issue 0.

[0072] Therefore, the objective of the present invention is to isolate mono expressed by the above hybridoma cells. The objective is to provide a clonal antibody that specifically recognizes 3pE Aβ. This can be done. Isolated monoclonal antibodies can be expressed by hybridoma cells, or It can be expressed by recombinant DNA.

[0073] Preferably, the antibody or antigen-binding fragment of the present invention selectively binds to 3pE Aβ, For other Aβ or β-amyloid precursor proteins (APP) that do not have 3pE There is little to no cross-reactivity. Specifically, the antibody or its antigenic antibody of the present invention. The combined fragment is Aβ 3pE-40 (SEQ ID NO: 40 or SEQ ID NO: 45) and Aβ 3pE-4 Aβ peptide that selectively binds to peptide 2 (SEQ ID NO: 51) and does not contain other 3pE molecules. There is little to no cross-reactivity with tide or APP.

[0074] Table 1 provides the amino acid sequences of the antibodies of the present invention. Kabat, Chothia, and The CDRs of the variable regions of the heavy and light chains, as defined by IMGT, are described as separate sequences. It 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 term "identity" or "percent identity" refers to the nucleic acid or polypeptide sequence when, for example, comparing an anti-3pE Aβ antibody and the polynucleotide encoding it, 3pE Aβ polypeptide and the 3pE Aβ polynucleotide encoding it, measured using one of the following sequence comparison algorithms or by visual inspection, and are the same when compared and aligned to maximize matches, or two or more sequences or subsequences having a specified percentage of the same amino acid residues or nucleotides. refers to.

[0086] For sequence comparison, typically, one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, and subsequence coordinates are specified if necessary, A rameter is designated. Next, the array comparison algorithm calculates the percentage of array identity of the test array relative to the reference array based on the designated program parameter.

[0087] Optimal alignment of the arrays for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat’l Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally, see Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0088] Examples of algorithms suitable for determining the percentage of array identity and array similarity are, respectively, Altschul et al. (1990) J. Mol. Biol. 215:4 ​​​​​​​​​​​​​​​ 03-410 and Altschul et al. (1997) Nucleic Aci BLAST and BL are described in ds Res.25:3389-3402 respectively. This is the AST2.0 algorithm. The software used for BLAST analysis is from the National Biotechnology Institute. Publicly available through the National Center for Biotechnology Information. It is possible. This algorithm first searches for words of the same length in the database array. When aligned, it matches or satisfies a threshold score T of some positive value. By identifying short words of length W in the query array, high This includes identifying score sequence pairs (HSPs). T is the adjacent word score threshold (Alts This is referred to as (chul et al., above). These initial adjacent word hits then It serves as a seed to initiate a search for longer HSPs that are present. Next, as long as the cumulative alignment score can be increased, work along each sequence Extend the hit in both directions.

[0089] Regarding nucleotide sequences, parameter M (reward for matching residue pairs) Uses Core (always > 0) and N (penalty score for mismatched residues, always < 0). Then calculate the cumulative score. For amino acid sequences, use a scoring matrix. Then calculate the cumulative score. The cumulative alignment score is lowered by X from its maximum score. When this occurs, the accumulation of alignment of one or more negative scoring residues results in When the cumulative score becomes zero or less, or when the end of any sequence is reached, each direction Stop the extension of word hits in BLAST algorithm. BLAST algorithm parameters W, T, And X determines the sensitivity and speed of the alignment. BLASTN program (Nucleo Regarding the CHIDO array, by default, the word length (W) is 11, the expected value is 10, and M=5. Use N=-4 and compare both chains. For amino acid sequences, see BLASTP Pro Gram defaults to a word length (W) of 3, an expected value (E) of 10, and BLOSUM. Use a 62-scoring matrix (Henikoff & Henikoff, Pr See oc.Natl.Acad.Sci.USA 89:10915(1989).

[0090] In addition to calculating sequence identity percentage, the BLAST algorithm uses two sequences 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)) is a combination of two nucleotides or amino acid sequences, where the matching occurs by chance. It provides an index of the probability of [something happening]. For example, nucleic acids provide an index of the minimum probability of [something happening] in a comparison between a test nucleic acid and a reference nucleic acid. The probability of success is less than about 0.1, more preferably less than about 0.01, and most preferably about 0.001. If it is less than [a certain value], it is considered similar to the reference array.

[0091] Further indicators that two nucleic acid sequences or polypeptides are substantially identical are described below. As shown, the polypeptide encoded by the first nucleic acid is coded by the second nucleic acid. It is immunologically reactive with the polypeptide being treated. Therefore, the polypeptide The chide is typically substantially identical to a second polypeptide, e.g., the two peptides differ only by conservative substitutions. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0092] In vitro methods Assays in which an antibody or an antigen-binding fragment thereof is bound to a solid phase, and assays in which an antibody is in a liquid medium All modes of immunoassay using an antibody or an antigen-binding fragment thereof, including an assay, should be understood to be contemplated for use in the presently preferred embodiments. Immunoassay methods that can be used to detect a sample using an antibody embodying the features of the present invention include competitive (reagent-limited) assays in which a labeled sample (sample analog) and the sample in the test sample compete for the antibody, and single-site immunoassay assays in which the antibody is labeled but are not limited thereto.

[0093] The antibody or antigen-binding fragment thereof according to the present invention can be used in conventional immunological techniques to detect Aβ3pE wherever it is present, including biological samples for monitoring β-amyloid-related diseases and conditioned media from cell cultures for monitoring intracellular processing of APP. Appropriate immunological techniques are well known to those skilled in the art and include, for example, ELISA, Western blot analysis, competitive or sandwich immunoassays, etc., all of which are well known to rely on the formation of antigen-antibody immune complexes, but here, for the purposes of the assay, the antibody or antigen-binding fragment thereof can be detectably labeled, for example, with a radioactive, enzymatic, luminescent or fluorescent label, or the antibody or antigen-binding fragment thereof The fragments can be immobilized on an insoluble carrier. Therefore, the object of the present invention is to remove A from the sample. The objective is to provide an immunoassay for the measurement or detection of β3pE or its fragments. The method involves, in accordance with the present invention, a sample having an antibody or its antigen-binding fragment being treated with Aβ3pE or its The fragment is brought into contact with the antibody or its antigen-binding fragment, and an immunocompound is formed between the antibody or its antigen-binding fragment and Aβ3pE or its fragment. This includes determining whether a body has formed. These methods involve tissue samples or body fluid samples. It can be done in either way, and generally involves obtaining a sample from the body of an individual, and then contrast-enhancing the sample. Contact with an effective amount of a detectably labeled antibody or its antigen-binding fragment according to the present invention. This includes detecting a label to establish the presence of Aβ3pE or a fragment thereof in the sample. The measurement method using the antibody or antigen-binding fragment thereof of the present invention is not particularly limited. In particular, antibodies, antigens, or antigens corresponding to the amount of Aβ3pE or its fragments in the solution being measured. The amount of antibody complex is detected by chemical or physical means, and a standard containing a known amount of antigen is used. Any measurement method may be used, as long as it is calculated from a standard curve prepared using the solution. For example, turbidimetric methods, competitive methods, immunoassays, and sandwich methods are preferably used. Regarding degree and specificity, the sandwich method is particularly preferred.

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

[0095] In the measurement method, labeling substances such as radiolabeled substances, enzymes, fluorescent substances, and luminescent substances are used as labeling agents. Examples of radiolabeled substances include 125 1, 131 I, 3 H and 14 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ It may be used. The recovered complex is directed towards the antigenic determinant on the antigen and binds to the antigen in the complex. It is brought into contact with at least one secondary antibody that can be combined. The secondary antibody is directed at the antibody The primary determinant is the antigenic determinant to which the primary antibody is directed due to the multiepitope property of the antigen entity. It is also acceptable to use either a primary or secondary antibody with any of the above labels. It may be made detectable. In a preferred embodiment, the secondary antibody is made detectable. Primary antibody The presence of a detectable antibody bound to a complex consisting of an antigen bound to a secondary antibody is a sign of the technology. It can be easily detected using techniques known in the field. It is obtained in biological samples. By comparing these results with those obtained from the control sample, the presence or absence of altered Aβ3pE can be determined. Its fragment level can be measured.

[0097] In vivo method Aspects of the present invention include preventing and improving amyloid-beta deposition in amyloid-beta-related conditions. Methods for treating and / or reducing, and for subjects requiring such treatment, as specified herein. A method comprising administering an antibody or its antigen-binding fragment disclosed herein in a therapeutically effective dose. Further aspects of the present invention include antibodies or antigen-binding fragments thereof disclosed herein. To prevent, improve, treat, and / or reduce amyloid deposition in myloid-beta-related conditions. The present invention comprises a pharmaceutical composition for the purpose of [details omitted]. The method of the present invention involves [details omitted] an effective amount of one of the following described herein. This includes administering the antibody or its antigen-binding fragment to a subject who requires it.

[0098] In one embodiment, the present invention relates to a puller containing β-amyloid protein in humans. Prevention, improvement, and treatment of amyloid-beta deposition in conditions characterized by the formation of cysts. Regarding methods for reducing, and / or reducing, the method relates to a therapeutically or prophylactically effective amount of human The antibody according to the present invention, which is an antibody that specifically binds to Aβ3pE, or the immunologically reactive antibody thereof. This includes administering fragments of the substance, preferably peripherally, to a person in need of such treatment. In another aspect, the present invention relates to a method for inhibiting the formation of amyloid plaques, and / or in humans Regarding a method for removing amyloid plaques, this method involves antibodies that remove Aβ3pE peptide in the brain. The effective amount according to the present invention isolates and induces altered Aβ3pE clearance in the brain. This includes administering antibodies to human subjects requiring such inhibition or removal. In some embodiments, the present invention relates to such humanized antibodies containing immunologically effective portions, and those Regarding methods for preparing [the product].

[0099] The target of this is the formation of plaques containing β-amyloid protein. A person who is suffering from or has a predisposition to the characteristic condition. One embodiment In this embodiment, the condition is Alzheimer's disease. In another embodiment, the condition is triso Mee-21 (Down syndrome), diffuse Lewy body dementia, inclusion body myositis, cerebral amyloid angiopathy, Or cognitive impairment associated with hereditary brain hemorrhage with Dutch type amyloidosis (HCHWA-D). It is a disease.

[0100] Humanized antibodies increase their similarity to antibody variants that are naturally produced in humans. These are antibodies derived from non-human species whose protein sequences have been modified. Generally speaking, humanized antibodies have modified proteins. The chromosome sequence is of non-human origin and is involved in the antibody's ability to bind to its target antigen. Except for some or all of the CDR segments, the protein sequence of the human variant and the original They are qualitatively identical. The variable domain framework domain corresponds to the human framework domain. It is replaced by and the non-human CDR remains virtually intact. In some cases Humanized antibodies, in order to maintain the binding affinity and / or dissociation constant of non-human antibodies, It has a small number of substitutions in one or more of the CDR regions.

[0101] Furthermore, humanized antibodies are derived from a human framework and at least one CDR from non-human antibodies. This refers to an antibody that contains and, in which any existing constant region is the human immunoglobulin constant region. They are substantially identical, that is, at least about 85%, 90%, preferably at least 95% are identical or 98% are identical. Therefore, all parts (one or more) of the humanized antibody are identical. (Excluding the CDR) is substantially identical to the corresponding portion of the human immunoglobulin sequence. For example, humanized immunoglobulins typically use chimeric mouse variable region / human constant region antibodies. It does not include.

[0102] Humanized antibodies are at least 3 times more effective than non-human and chimeric antibodies for use in the treatment of humans. It has several potential advantages: 1) Since the effector part is human, it is not related to other parts of the human immune system. It can interact better with (for example, by activating microglia and plaque) 1) Removes the foreign substance. 2) The human immune system recognizes the framework or C region of humanized antibodies as foreign. Since they shouldn't be aware of it, the antibody response to such administered antibodies is entirely due to the foreign non-hybrid. 3) The response should be lower than that to the administered non- Human antibodies have been reported to have a shorter half-life in the human circulation than the half-life of human antibodies. It is.

[0103] To treat or prevent a condition characterized by the formation of plaques containing β-amyloid protein. In a method for this purpose, the antibody of the present invention or its antigen-binding fragment (containing an immunologically reactive fragment) (mu) refers to Alzheimer's disease, dementia associated with Down syndrome, and dementia before the clinical or symptomatic onset of symptoms. This is a risk factor for amyloid-beta-related symptoms or conditions such as amyloid angiopathy, which may be clinical or pre-symptomatic. It is administered to individuals that are aggressive or exhibit aggressive tendencies using standard administration techniques. Preferably It is administered peripherally (i.e., not to the central nervous system), intravenously, intraperitoneally, subcutaneously, and in the lungs. It is administered via transdermal, intramuscular, intranasal, intrachungal, sublingual, or suppository administration. Antibodies or their binding The fragments may be administered directly to the ventricular system, cerebrospinal fluid, or brain parenchyma, and techniques directed to these locations are Although these methods are well known in the relevant field, there is no need to use these more complex techniques. The antibody or its conjugated fragment is effective when administered by a simpler technique involving the peripheral circulatory system. It is effective. The advantage of the present invention is that even if antibodies or other substances are not directly provided to the central nervous system itself, they are effective. The antigen-binding fragment possesses the ability to exert its beneficial effects.

[0104] The pharmaceutical composition for administration is designed to be suitable for the selected mode of administration, and contains a dispersant, a stimulant, and a stimulant. Medicinal excipients such as abutments, surfactants, preservatives, solubilizers, isotonic agents, and stabilizers. It is used when appropriate. Remington's is incorporated herein by reference. s Pharmaceutical Sciences,Mack Publishing g Co., Easton Pa., latest edition is generally for experts. This provides an overview of known prescription techniques.

[0105] For example, by encapsulating them in liposomes, or by blocking polar groups. Therefore, making them more lipophilic and changing the solubility characteristics of the antibody of the present invention is particularly important. It may be useful.

[0106] Peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection is preferred. A suitable vehicle for this is simple. However, administration can also be done via nasal aerosol or suppository. The drug may be administered through the mucous membrane. Formulations suitable for such administration methods are well known. Typically, these include surfactants that promote intermembrane transport. Such surfactants are used in many applications. The compound is derived from steroids, or N-[1-(2,3-dioleoyl)propyl-N Cationic lipids such as N,N-trimethylammonium chloride (DOTMA), or Various compounds such as cholesterol hemisuccinate and phosphatidylglycerol ru.

[0107] The concentration of humanized antibodies in the formulation ranges from only about 0.1% to about 15 or 20% by weight, mainly through flow. Based on body volume, viscosity, etc., it is selected according to the specific dosage regimen chosen. Therefore, note A typical pharmaceutical composition for injection is 1 mL of sterile buffer water of phosphate-buffered saline, and 1-1 The formulation can be prepared to contain 00 mg of the humanized antibody of the present invention. It may be subsequently sterile filtered or otherwise made microbiologically acceptable. Typical compositions for internal injection include 250 mL of fluid, for example, the volume of sterile Ringer's solution, and It may have an antibody concentration of 1 to 100 mg / mL or more per 1 mL.

[0108] Regarding antibody administration, the dosage is approximately 0.0001 to 100 mg / m³ relative to the host's body weight. The dose is in the range of 0.01 to 75 mg / kg, preferably in the range of 0.01 to 75 mg / kg. For example, the dose is the host's body The dosages are 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, and 0.75 mg / kg per unit of weight. 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 terms of administration, the dosage is within the range of 0.01 to 10 mg / kg, or 0.1 to 15 mg Within the range of / kg, or within the range of 0.1 to 20 mg / kg, or 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, less At least 5 mg / kg, at least 10 mg / kg, at least 20 mg / kg, less 30 mg / kg, at least 40 mg / kg, at least 50 mg / kg, or less The minimum dose is 60 mg / kg. In preferred cases, the dose is approximately 10 kg / mg, approximately 20 kg g / mg, approx. 30kg / mg, approx. 40mg / kg, approx. 50mg / kg, approx. 60mg / kg , or it may be about 70 mg / kg. In a particularly preferred example, the antibody may be about 0.3 mg / kg It is administered intraperitoneally in a dose range of approximately 60 mg / kg. In exemplary treatment regimens, The body is approximately 10 kg / mg, approximately 20 kg / mg, approximately 30 kg / mg, approximately 40 mg / kg, approximately It is administered intraperitoneally at a dose of 50 mg / kg or approximately 60 mg / kg.

[0109] As used herein, the term "approximately" when used in relation to measurable values ​​such as quantities refers to the same thing. ±20% to ±0.1%, preferably ±15% or ±10%, and ±5% of the civil standard. This includes variations of ±1% for comfort, ±0.5% for even more comfort, and ±0.1% for further comfort. This means that a variation of ±0.05% or ±0.01% from a specific value is That is appropriate.

[0110] A typical treatment regimen involves administration every two weeks, once a month, or once every three to six months. This is accompanied by two or more monoclonal antibodies with different binding specificities. They are administered simultaneously, and the dosage of each antibody administered is within the prescribed range. The body is usually administered in multiple doses. The interval between doses may be weekly, monthly, or It can occur annually. The interval can also be irregular, which is the resistance to Aβ in the subject. This is indicated by measuring the blood level in the body. Alternatively, antibodies are available as sustained-release formulations. It can be administered, and in this case, the frequency of administration required will be reduced. The dosage and frequency will depend on the patient. It varies depending on the half-life of the antibody in the individual. 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 preventive or curative. For preventative use, relatively low doses are administered at relatively infrequent intervals over a long period of time. It is administered standing. Some patients continue treatment for life. For therapeutic purposes, Preferably, until the progression of the disease decreases or stops, and the subject experiences partial or complete relief of the disease symptoms. It may be necessary to administer relatively high doses at relatively short intervals until a solution is found. After that, prophylactic treatment Dimen may be administered.

[0112] In some methods, the dosage is determined so that the plasma antibody concentration is approximately 1-1000 μg / mL. In addition, in some methods, it is administered at a concentration of approximately 25-300 μg / mL. Alternatively, antibodies can be administered as sustained-release formulations, in which case the required frequency of administration is The dosage will decrease. The dosage and frequency will vary depending on the half-life of the antibody in the target population.

[0113] Therapy with the antibody of the present invention may be performed as monotherapy. Alternatively, the antibody of the present invention may be used as... The treatment is one of a combination therapy regimens that also uses one or more additional medications to treat the individual. It may also be a component or a stage.

[0114] When used in vivo, the antibody or antigen-binding fragment of the present invention is used in a therapeutically effective amount. For example, reducing, removing, or preventing β-amyloid plaques, or AD or Administered to individuals in doses that improve cognitive function in subjects with other β-amyloid-related diseases. The antibody or its antigen-binding fragment is administered intravenously, for example, as a bolus or over a period of time. Through continuous injection over a period of time, it can be administered intramuscularly, intraperitoneally, intracerebrospinal fluid, subcutaneously, intra-articularly, intra-synovially, and intrathecally. The present invention is administered to an individual by known methods, such as by oral, topical, or inhalation routes. These drugs are, if desired, at least partially effective in treating amyloidogenic diseases. It can be administered in combination with the following drugs: Alzheimer's disease and amyloid deposits in the brain In the event of the associated condition, the antibody or antigen-binding fragment of the present invention crosses the blood-brain barrier. It may be administered in combination with other drugs that increase the passage of certain medications.

[0115] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof in plaque deposits It binds to 3pE Aβ. By binding to 3pE Aβ in plaque deposits, the antibody Alternatively, the antigen-binding fragment can induce plaque removal. The process involves the activation of microglue surrounding the plaque and the removal of stable Aβ morphology. This may be due to the destabilization of the marker. Furthermore, the antibody or antigen-binding fragment of the present invention is It may prevent the plaque-seeding activity of 3pE Aβ. Compared to vascular amyloid, The potential concentration of 3pE Aβ in the drug increases the range of therapeutic safety for immunotherapy. It is possible.

[0116] Kits and devices The present invention provides kits and devices that can be used in the manner described above. The kit and device include an antibody or antigen-binding fragment thereof that binds to 3pE Aβ. Furthermore, the kit may include reagents and instructions. The instructions may be printed on paper, for example. It may be provided in an electronically readable medium, or in an instruction manual. For example, an internet web server designated by the kit manufacturer or distributor. It may be provided by guiding the user to a website.

[0117] The reagents included in the kit of the present invention are substantially adsorbed or altered by the container material. While not being altered, all kinds of containers are designed so that the activity of various components is not substantially lost. It can be supplied by [company name].

[0118] In one embodiment, the kit or device comprises the antibody of the present invention or its antigen-binding fragment, More preferably purified antibodies, more preferably monoclonal antibodies, and even more preferably 3p antibodies. The present invention contains an isolated monoclonal antibody that binds to the E Aβ peptide. In the embodiment, the antibody is It is expressed by hybridoma cells.

[0119] Embodiment Embodiment 1 is, a. Sequence numbers 1, 2, 3, 4, 5, and 6, respectively b. Sequence numbers 1, 7, 3, 4, 5, and 6, respectively c. Sequence numbers 1, 7, 3, 8, 5, and 6, respectively d. Sequence numbers 1, 2, 3, 8, 5, and 6, respectively e. Sequence numbers 56, 57, 3, 8, 5, and 6, respectively. f. Sequence numbers 56, 57, 3, 4, 5, and 6, respectively. g. Sequence IDs 56, 58, 3, 4, 5, and 6, respectively h. Sequence numbers 56, 7, 3, 8, 5, and 6, respectively. i. Sequence numbers 1, 57, 3, 8, 5, and 6, respectively j. Sequence numbers 56, 7, 3, 4, 5, and 6, respectively. k. Sequence numbers 1, 57, 3, 4, 5, and 6, respectively. l. Sequence IDs 1, 58, 3, 4, 5, and 6, respectively, or m. Each has the polypeptide sequences of sequence numbers 56, 2, 3, 4, 5, and 6, respectively. , Multicomplementarity Determination Region 1 (HCDR1), HCDR2, HCDR3, Light Chain Complementarity Determination Region 1 (LCDR1), LCDR2, and LCDR3 isolated monoclonal antibodies or their anti- The protoconjugated fragment is an antibody or its antigen-binding fragment, preferably 3pE Aβ, and more preferably human 3p E is an isolated monoclonal antibody or its antigen-binding fragment that specifically binds to Aβ.

[0120] Embodiment 2 is represented by sequence numbers 9, 11, 13, 15, 16, 17, 19, 20, or 2 A heavy chain variable region having a polypeptide sequence that is at least 95% identical to 1, or SEQ ID NO: 10 , 12, 14, 18, 22, 53, or 55 are at least 95% identical polypeptides. An isolated monoclonal antibody according to Embodiment 1, comprising a light chain variable region having a sequence, or the same It is an antigen-binding fragment.

[0121] Embodiment 3 is, a. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly Light chain variable region having a peptide sequence, b. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and the polypeptide sequence of SEQ ID NO: 10 Light chain variable region having a ptide sequence, c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11 and the poly Light chain variable region having a peptide sequence, d. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13 and the poly Light chain variable region having a peptide sequence, e. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and the poly Light chain variable region having a peptide sequence, f. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 16 and the poly Light chain variable region having a peptide sequence, g. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 20 and the poly Light chain variable region having a peptide sequence, h. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17 and the poly Light chain variable region having a peptide sequence, i. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19 and the poly chain of SEQ ID NO: 18 Light chain variable region having a peptide sequence, j. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly A light chain variable region having a peptide sequence, or k. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and the poly The isolated monoclonal antibody according to Embodiment 1, comprising a light chain variable region having a peptide sequence. Or it is an antigen-binding fragment thereof.

[0122] Embodiment 4 is an embodiment in which the isolated monoclonal antibody or its antigen-binding fragment is a chimeric form It is an isolated monoclonal antibody or its antigen-binding fragment described in any one of the three states 1 to 3.

[0123] Embodiment 5 is a case where the isolated monoclonal antibody or its antigen-binding fragment is human or humanized. The isolated monoclonal antibody or its antibody described in any one of Embodiments 1 to 4. This is the original binding fragment.

[0124] Embodiment 6 is, a. Heavy chain amino acid sequence including SEQ ID NO: 37 and light chain amino acid sequence including SEQ ID NO: 38 , b. Heavy chain amino acid sequence containing SEQ ID NO: 39 and light chain amino acid sequence containing SEQ ID NO: 38 , c. Heavy chain amino acid sequence including SEQ ID NO: 37 and light chain amino acid sequence including SEQ ID NO: 52 , or d. Heavy chain amino acid sequence including SEQ ID NO: 39 and light chain amino acid sequence including SEQ ID NO: 54 It is an isolated monoclonal antibody containing [the specified substance].

[0125] Embodiment 7 is a monoclonal antibody or its antibody described in any one of Embodiments 1 to 6. This is an isolated nucleic acid encoding the original binding fragment.

[0126] Embodiment 8 is a vector containing the isolated nucleic acid described in Embodiment 7.

[0127] Embodiment 9 is a host cell containing the vector described in Embodiment 8.

[0128] Embodiment 10 is an isolated monoclonal antibody described in any one of Embodiments 1 to 6 or The pharmaceutical composition comprises the antigen-binding fragment and a pharmaceutically acceptable carrier.

[0129] Embodiment 11 contains β-amyloid protein in the target where it is needed. A method for treating a condition related to plaque formation, the method is one of embodiments 1 to 6 A monoclonal antibody or its antigen-binding fragment as described in any one of the following, or as described in Embodiment 10. The method involves administering the listed pharmaceutical composition to a target subject requiring its use.

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

[0131] Embodiment 13 describes a condition in which the patient has dementia associated with trisomy 21 (Down syndrome), and diffuse symptoms. Lewy body dementia, inclusion body myositis, cerebral amyloid angiopathy, and Dutch-type amyloidosis. The method according to Embodiment 11, selected from the group consisting of hereditary cerebral hemorrhage (HCHWA-D). That is the case.

[0132] Embodiment 14 describes a plaque associated with Alzheimer's disease in a subject that requires it. A method for reducing the monoclonity, the method being described in any one of Embodiments 1 to 6. A nal antibody or its antigen-binding fragment, or the pharmaceutical composition described in Embodiment 10, is required. This method includes administering the substance to the target.

[0133] Embodiment 15 prevents the seeding activity of 3pE Aβ in subjects where it is needed. A method for stopping a monoclonal anti- A body or its antigen-binding fragment, or the pharmaceutical composition described in Embodiment 10, is required by the target. This method includes administering it to [the target].

[0134] Embodiment 16 is a monoclonal antibody or the same described in any one of Embodiments 1 to 6. A method for generating an antigen-binding fragment, comprising generating a monoclonal antibody or its antigen-binding fragment. Under these conditions, a fragment containing nucleic acids encoding a monoclonal antibody or its antigen-binding fragment. A method comprising culturing cells and recovering antibodies or their antigen-binding fragments. .

[0135] Embodiment 17 is a monoclonal antibody or the same described in any one of Embodiments 1 to 6. A method for producing a pharmaceutical composition containing an antigen-binding fragment, wherein the method involves a monoclonal antibody or The antigen-binding fragment is combined with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition. It includes methods. [Examples]

[0136] The present invention can be further understood by considering the following non-limiting embodiments.

[0137] Example 1: Production and humanization process of monoclonal antibodies Three Balb / c mice (Janvier Labs) were subjected to complete Freund's ajuga treatment. H2N-pEFRHDSGC-CO in Sigma (St. Louis, MO) Initial immunization was performed with OH(Eurogentec) (SEQ ID NO: 47). COOH-terminal cyst Inject Maleimide A via the yin residue, according to the manufacturer's instructions. Certified BSA kit (Pierce (Rockford, IL)) and other cities Using a commercially available kit, maleimide-activated bovine serum albumin (Life Techno By coupling peptides to logies (Carlsbad, CA), Peptides were prepared. Mice were first given a complete Freund's adjuvant, followed by an incomplete furthone. Coupling of 100 μg or 200 μg of BSA in the hemoglobin adjuvant (Sigma) Additional immunizations were administered every two weeks using the peptide formulation.

[0138] Hybridoma and antibody production: Select mice with the highest serum titer for fusion. Meanwhile, the spleens of other mice were isolated and frozen in liquid nitrogen. On day 4, fusion or spleen Before excision, all mice were given BSA (Merck (Kenilworth) in physiological saline. , 100 μg of H2N-pEFRHDSGC-COOH coupled to NJ) Mouse splenocytes were immunized intraperitoneally with Sequence ID No. 47. The modification procedure of tein (Euro.J.Immunol.,1976;292-295) Therefore, it was fused with SP2 / 0 cells (ATCC (Manassas, VA)). Doma was seeded into a 30x96 well plate, and after 10 days, 0.5 μg / well was administered. Uncoupled Aβ 3pE-40 peptide (AnaSpec(Fremon) t, CA)) were screened directly by ELISA. Positive cells were treated with 0.5 μg / mL Cross-reactivity (lack of cross-reactivity) with coated Aβ1-40 peptide (AnaSpec) We tested it and immediately performed subcloning.

[0139] After fusion, 17 clones used human Aβ3pE-40 synthetic peptide (SEQ ID NO: 40). In ELISA screening, which is directly coated, it reacts positively, and the liquid It was frozen in nitrogen.

[0140] All hybridomas were tested using 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 The cells were cultured in Dulbecco's modified Eagle medium supplemented with treptomycin (50 mg / mL). All products are commercially available and were purchased from Life Technologies. The cells were incubated in a humidified 8% CO2 air incubator.

[0141] Direct ELISA for antibody selection: The same method used to detect the Aβ 3pE-40 antibody described above. The cleaning ELISA uses 0.5 μg / mL of free human Aβ 3pE-40 peptide. (SEQ ID NO: 40) was incubated overnight at 4°C in 50 μL / well of coating buffer (10 mM T). NUNC Ma in ris, 10 mM NaCl and 10 mM NaN3, pH 8.5) xisorp(Life Technologies) flat bottom high coupling 96 well microtaper This was a direct ELISA using an ELISA coated on an ether plate.

[0142] The following day, 0.1% casein (Merck) in 75 μL / well PBS was incubated at room temperature for 60 minutes. The plate was then blocked to reduce nonspecific binding. Next, 50 μL of hybrid The doma supernatant was added and incubated at 37°C for 1 hour. After washing, the conjugated monoclonal The Naru antibody was incubated at 37°C for 1 hour in 50 μL / well of horseradish peroxidase (Ame). rsham-Pharmacia Biotech(Little Chalfont, It was detected using sheep anti-mouse IgG complexed with United Kingdom. The reagent was diluted with 0.1% casein / PBS. The plate was washed, and 50 μL of 0.42 3,5,3',5'-tetramethylbenzidine (Biorad) at mM, 0.003% (Volume / Volume) of H2O2 (Biorad) (100mM citric acid (Biorad(H Ercules, CA)) in 100 mM disodium hydrogen phosphate (pH 4.3) A solution of Biorad was added as a substrate. The mixture was left on a plate shaker at room temperature for up to 15 minutes. Allow the intermolecular reaction to proceed, and then develop the color with 50 μL / well of 2N H2SO4 (Merck). The system was stopped, and the plate was read at 450 nm using a microtiter plate reader. (Thermomax, Molecular Devices). Selected monoclonal Cross-reactivity between the antibody and full-size human free Aβ 1-40 was observed in the screening assay. The test was performed using the same direct ELISA method as in (I).

[0143] From 17 Aβ3pE-40-reactive clones, further selection was made based on affinity and selectivity. BAMB31_1 was selected for characterization (see Examples 2 and 3). This antibody It was determined that it possesses the mouse IgG1 isotype heavy chain and mouse κ light chain. Mouse I gG1 Fc has 70% sequence identity and 76% sequence identity with mouse IgG2a Fc. Although they share only similarities, these isotypes have different activity and protein profiles. It has a characteristic. Compared to mouse IgG2a, mouse IgG1 has mouse FcγRI, Fc Due to weaker binding to γRIII and FcγRIV receptors and mouse C1q, It has weak Fc effector and complement function. It is the isotype closest to human IgG1 activity. Therefore, mouse IgG2a is thought to be mouse FcγRI, FcγRIII, and Fc By binding to the γRIV receptor and mouse C1q, Aβ plaque clearance is achieved. Complement and antibody-dependent cellular cytotoxicity may contribute to the development of the immune system. ty, ADCC), and antibody-dependent cellular phagocytosis (antibody-dependent cellular phagocytosis). It has ADCP activity.

[0144] The BAMB31 heavy chain sequence was changed from mouse IgG1 to mouse IgG2a, and BAM B31_2a was generated. The reactivity after cloning the V region was preserved using the SPR described below. This was confirmed by law.

[0145] Humanization process: For this work, AbM definition (Martin, AC, PNAS8) Except for CDR-H2 delineated according to 6:9268-9277, 1989), Si Use the same procedure as ngh, et al (MAbs2015;7(4):778-91). The parent antibody BAMB31_2a (mIgG2a) was then humanized. In short, complementarity Determinative regions (CDRs) were identified in the mouse parent sequence. These sequences were then used in the human germline and We compared it with four human germline heavy chains and selected two human germline light chain frameworks. Mouse CDRs were ported to the framework. Mouse J-segment sequences and human J-segment sequences. By comparing sequences, we select the human J segments for VL and VH of each parent antibody. This maximized sequence identity. The molecular model of the Fv region of the parent mAb was set to default parameters. This was generated using MOE (CCG (Montreal, Canada)) with the TA feature. The obtained model is examined graphically to identify potential factors affecting binding and / or antibody stability. Key framework locations were identified. These locations are in addition to the transplanted CDR, in addition to human / A mouse binary antibody library was created. In the library, transplanted Each strand containing the mouse CDR was paired with the strand of the mouse parent on the opposite side. In this way, one Only the chain is fitted to the human framework, and reverse mutations are determined based on antigen binding. Next, humanized VH and VL were combined to arrive at the final antibody candidate.

[0146] Library clones were expressed as Fab in E. coli and compared to the complete mouse parent molecule. The binding to the peptide was tested using signaling and ELISA. 80% of the mouse parent Molecular signals showing binding exceeding % were selected for sequencing. The sequences were analyzed, and Select and combine human-compatible heavy chains and human-compatible light chains to produce monoclonal human IgG1 The antibodies were expressed. All VH / VL humanized pairs of each antibody were expressed, purified, and antigenic compounds were formed. In addition, the immunogenetic risk of Epivax in silico is reverse mutation in the mouse sequence. The number of residues and their biophysical properties were evaluated.

[0147] Results: In order to maintain parental binding, it was necessary to revert some residues to the mouse sequence. The maintenance of the bond, the immunogenetic risks of Epivax in silico, and the organism The physical properties did not depend on the number of revert mutations in the mouse sequence, but their location was independent of the mouse sequence. The mutation was reversed. The results of the characterization of representative HFA mAbs from this analysis are shown in Tables 2 and 3. This will be shown.

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

[0149] Mitigation of post-translational modification risk: The parent antibody and human framework-adapted variants are HCDR2 It contained a post-translational deamidation modification motif in the NG. To address this potential problem... To do this, we use condensed oligonucleotides to create individual libraries for both N and G residues. It was done. These newly created sequences are randomly assigned 20 meshes to each position. No acids were introduced. Each library was screened for retained binding. (Mouse) Mutant antibodies that exhibited similar binding to the parent antibody were selected for sequencing.

[0150] Results: After sequencing, both the N~S and N~G mutants exhibited binding comparable to that of the parent. The lead HFA mutant was cloned and expressed as wild-type IgG1 (BAMAB674). This results in IgG1 (Hc) having M37Y, S39T, and T41E point mutations in the Fc region. The constant region Fc IgG1 heavy chain sequence is based on GenBank accession number AEV43323. The mutation (numbered) was named +YTE IgG1(BAMB675). These mutations are F It is known to increase affinity for cRn and increase the 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 characteristics of 74 and BAMB675 are shown in Table 3.

[0151] [Table 3]

[0152] [Table 4] * N=1

[0153] Example 2: Thermal Stability Test Regarding the BAMB31 human framework-adapted mutant, these thermal stabilities were demonstrated using nanotechnology. We use nano differential scanning fluorimetry (NanoDSF). The melting onset temperature (Tonset), the first melting transition temperature (Tm1), and the initial melting transition temperature were evaluated. The temperature for detecting agglutination (Tagg) was measured. Some of the mutant data are shown in Table 2 (columns 6-8). This is shown in Table 3 (rows 10-12) and Table 3a.

[0154] Materials and Methods: The thermal stability of the sample was determined using an automated Prometheus instrument. The measurement involves loading the sample from a 384-well sample plate into a 24-well capillary tube. The procedure is as follows: Each sample is measured twice. Prometheus Nano To perform the procedure, use the DSF user interface (Melting Scan tab). Set the experimental parameters. Thermal scanning of a typical IgG sample is performed at a rate of 1.0°C / min. The temperature range is 20°C to 95°C. Typical sample concentrations are in the range of 0.3 to 1 mg / mL. 3 Using molecular-specific fluorescence at 30 nm and 350 nm, an amphora in a temperature lamp is used. The loading is monitored and recorded as a change in fluorescence intensity over time. This is the result of thermal scanning. It is called (Tm). In parallel, using back reflection technology, the equipment heats up The start of aggregation (Tagg) in the sample is calculated. Therefore, the NanoDSF method is used under various conditions. The three-dimensional structure of lead candidates is often monitored as an indicator of long-term sample stability under the following circumstances. This also enables simultaneous measurement of colloidal stability.

[0155] Example 3: Immunogenetic risk assessment of Epivax in silico EpiMatrix software (EpiV) for predicting MHC class II binding. Using ax Inc., in silico fraction of the V region of anti-Aβ 3pE mab Analysis was performed. The software examined consecutive amino acid 9-mers to determine the potential HLA class. Identify II-binding sequences. The database is the most common H, representing approximately 95% of the human population. Includes LA type. The site where HLA receptors on antigen-presenting cells bind to peptide agretope. In addition, the other side of the peptide (epitope) binds to T effector cells or T regulatory cells. This can stimulate an immune response against the protein that has that epitope. Or it can lead to suppression. The software is for predicting the binding of T regulatory cells. It generates an agretope binding score that can be adjusted. The score is the protein's The number of binding events that lead to an output indicating the predicted immunogenicity of IZ and proteins is normalized. It can be done.

[0156] Example 4: Analytical characteristics of purified mAbs The protein concentration of each purified mAb was measured using a NanoDrop1000 spectrophotometer or T Absorbance at 280 nm using a rinean DropSense96 multi-channel spectrophotometer. The degree was determined by measuring it, and then calculated using a decay coefficient based on the amino acid sequence.

[0157] 0.2MN measured at 1 mL / min for 20 minutes on Waters Alliance HPLC. At a phosphate pH of 6.8, TOSOH TSKgel BioAs SE of antibodies purified by running the sample on the sist G3SWxl column HPLC was performed. The column eluate was monitored by absorbance at 280 nm. Results This is shown in Tables 2 and 3.

[0158] Example 5: Measurement of binding dynamics and affinity Surface plasmon resonance (SPR) is used to study biomolecular interactions. This is a label-free detection method used for investigation. It detects small changes in mass on the sensor surface. By monitoring, this direct real-time binding assay provides qualitative information about interactions between biomolecules. Provides objective and quantitative data; namely, equilibrium coupling constant (affinity, K D ) and dynamic constants (ka / k d ; Association rate of the complex k a , and the dissociation rate of the complex k d ) will be decided. The law addresses protein-protein and protein-nucleic acid interactions, as well as protein- and small It is useful in studying interactions with molecules. Here, 3pE-specific antibodies and human Aβ3 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 We investigated the interaction with β3pE-28 peptides (SEQ ID NOs. 45 and 46).

[0159] Materials and methods SPR:GE Healthcare mouse antibody capture kit, Aβ-3pE-4 Affinity test of BAMB31_2a (mIgG2a) against peptide 0 (SEQ ID NO: 40) Used in (as described in U.S. Patent Application Publication No. 2018 / 0142011) J&JP RD / Aβ / pE3 / 1 mIgG2a and (US Patent No. 9,944,696 and US Patent) This product contains mE8c mIgG2a (as described in publication No. 8679498) as a control antibody. According to the manufacturer's protocol, anti-maturation is transmitted via amine coupling on the CM5 sensor chip. The antibody was immobilized. Subsequently, the target antibody (1 μg / mL) was mixed with anti-mouse antibody. After capturing the substance to a concentration of 300 RU, the running buffer (2.7 mM KCl) is then added. , 137 mM NaCl and 0.05% surfactant P20 (Tween(trademark) 20) Various concentrations (3.125 nM, 6.25 nM, 6.25 nM) diluted with 20 mM phosphate buffer containing [the substance]. Human Aβ 3pE-40 peptide (12.5 nM, 25 nM, and 50 nM) (SEQ ID NO: 4) 0) was injected. The surface was treated with 10 mM glycine HCl at pH 1.7 for at least 180 seconds. The sample was then regenerated for another 60 seconds. Human Aβ(1-40) peptide (SEQ ID NO: 41) was used as a negative control. I used it.

[0160] Affinity measurement is performed using the optical biosensor T200 (Biacore®). I went. Biacore T200 Evaluation Software (version Using version 2.0), dynamic analysis was performed according to a 1:1 coupled fitting model.

[0161] In some cases, the binding affinity and specificity of anti-Aβ 3pE mAbs differ depending on the instrument (B iacore T200, Biacore 8K or MASS-2 (Biacore, I (nc.)) and performed using anti-human or anti-mouse immunoglobulin biosensor surfaces. Measured by SPR. Anti-human or anti-mouse immunoglobulin antibodies were detected by amine-cutting. Use the manufacturer's instructions for the CM4 sensor chip or CM5 sensor from Pulling Chemicals. The target antibody was covalently bonded to the surface of a chip (GE Healthcare). Alternatively, after capturing the immunoglobulin to a concentration of 300-400 RU on a mouse immunoglobulin sensor chip, 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 fibr Contains 0.005% surfactant P20 (Tween® 20) from Ronectin. Inject at various concentrations in HEPES-buffered saline. 30 μL at 100 μL / min The surface was regenerated by injecting 10 mM Gly pH 1.5 in two pulses. The reported data was captured. This refers to the difference in SPR signaling between fluid cells containing antibodies and reference cells that do not contain capture antibodies. The data for blank injection is obtained from the reference-subtracted signal. By subtraction, the additional contribution of the instrument to the signal was removed. Applicable. In this case, use Biaevaluation software (Biacore, Inc.) The association and dissociation phases at all concentrations (global fit) are fitted into a 1:1 binding model. The data was analyzed by applying the settings. If not applicable, the data was combined. The presence / absence of the bond was evaluated qualitatively.

[0162] Immunohistochemical analysis of formalin-fixed, paraffin-embedded brain: For immunohistochemical analysis Therefore, after deparaffinizing and rehydrating the sections, they were subjected to thawing in formic acid (70% distilled water) for 10 minutes. Antigen retrieval was performed by incubating brain slides from mutant mice. Causative peroxidase activity is reduced by 3% hydrogen peroxide (DAKO; (Glostrup, Denm Blocked by ark)S2023). Background reduction component (DAKO, Different concentrations (active concentration: 2, 0.1-0.05-0) in antibody diluents having S3022. (0.025 μg / mL), BAMB674, BAMB675, hE8L, R17L, R1 7. CI-C7, B12L, Antibody I or Antibody II (The following seven antibodies are U.S. 99 As stated above in U.S. Patent No. 44696(B2) and U.S. Patent No. 8679498(B2) The sections were incubated with ) for 1 hour. After extensive washing, HRP-labeled anti-human secondary Antibody (Antibody diluent (DAKO, S0809) PI-3000 (Vector l Apply abs(Burlingame,CA)-1 / 500) to the slide for 1 hour. Next, 3,3-diaminobenzidine (DAB) (DAKO, K3468) is used for colorimetric labeling. I did it. I counterstained the slides with hematoxylin, dehydrated them, and then performed a Vectormount (H- It was permanently fixed with a 5000 (Vector Labs) device.

[0163] Results: Dynamic analysis of the monoclonal antibody BAMB31_2a (mIgG2a) showed that A Affinity binding to β3pE-40 peptide (SEQ ID NO: 40) was confirmed. Maximum binding is 50n. When human Aβ-140 peptide (SEQ ID NO: 41) was applied at concentration M, binding was not detected. It was not possible. BAMB31_2 for human Aβ3pE-40 peptide (SEQ ID NO: 40) Figure 1 shows a sensorgram (single-cycle dynamics) illustrating the binding interaction of a(mIgG2a). As shown below, the molecules used as the comparison standard are J&JPRD / Aβ / pE3 / 1 mIgG2a and mE8c and mIgG2a (Figure 2) were evaluated using the same assay, and mE8c and J&JPRD were evaluated. It was demonstrated that BAMB31 has a higher affinity compared to / Aβ / pE3 / 1. Equilibrium binding. constant (affinity, K D ) and dynamic constant (k a / k d The results are shown in Table 4.

[0164] [Table 5]

[0165] BAMB31_2a (mIgG2a) parent and BAMB246 (human IgG1 chimera) parent In comparison, BAMB31 HFA has a reduced risk of Hc CDR2 NG deamidation. The kinetic analysis of mAbs (BAMB674 and BAMB675) was performed using Aβ3pE-28 peptide. It was shown to retain binding affinity to tide (sequence number 42). Equilibrium binding constant (affinity) sex, K D ) and dynamic constant (k a / k d ) are shown in Table 5.

[0166] The aforementioned humanized 3pE-specific antibodies hE8L, R17L, R17, CI-C7, and B12L , Antibody I, and Antibody II (U.S. Patent No. 9944696(B2) and U.S. Patent No. 8679) As shown in Table 5, the current BAMB3 (as previously described in issue 498(B2)) 1. The affinity for HFA molecules is higher.

[0167] [Table 6] Human chimeric antibodies have a mouse variable region on top of the human IgG1 constant region.

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

[0169] The higher affinity measured by SPR also translates to improved plaque binding. The primary antibody is used to test on brain sections of transgenic mouse brains by immunohistochemistry. A dilution series was performed. All antibodies were in different ranges, but at 2 μg / mL, they were visible. Lark labeling is applied. Antibodies CI-C7 and R17L are administered at a concentration of 0.1 μg / mL. According to the low affinity measured by SPR, it does not produce a visible plaque label. At a concentration of 0.05 μg / mL, BAMB674 and BAMB675 were found to be effective against plaque. While it shows recognition, the plaque label is invisible at this concentration relative to the molecule used as a reference (Figure). 3) At a concentration of 0.025 μg / mL, visible plaque labeling was detected in all molecules tested. It is (almost completely) invisible.

[0170] In conclusion, the immunohistochemical data showed that BAMB674 and the reference molecule were comparable. We have confirmed SPR data showing higher affinity for BAMB675.

[0171] BAMB246 (human IgG1 chimera) and the current BAMB31 HFA mAb(B AMB674 and BAMB675 are highly homologous mouse Aβ 3pE-28 peptides. Selectivity more than 3 logs greater than (SEQ ID NO: 46), and for fibronectin, Aβ 1-28 Aβ 3pE-2 having peptide (SEQ ID NO: 43) and scrambled amino acids 3-9 8 peptide (scrambled 3pE-28) (SEQ ID NO: 44) exhibits selectivity more than 5 logs greater. It possessed the selectivity for related peptides and proteins and the equilibrium binding constant (affinity). sex, K D ) and dynamic constant (k a and k d ) are shown in Table 6.

[0172] [Table 8] Scrambled 3pE-28 is human Aβ 3pE having scrambled amino acids 3-9. It is a peptide of -28. The selectivity multiplier was calculated by dividing the KD in Table 4 by the KD in Table 3. ≥1.20E-06 K D In (M), antigen binding occurred up to a maximum concentration of 1.2 μM tested. Not detected.

[0173] Wild-type IgG1 (BAMB674) and +YTE IgG1 isotype (BAMB6 Table 7 shows the FcRn binding affinity of BAMB31 HFA antibody against 75). +YTE mAb (BAMB675) was found to be effective in pharmacokinetic studies of cynomolgus monkeys with +YTE antibody Compared to wild-type IgG1 (BAMB674) converted to have a longer circulating half-life, human and It has approximately three times higher affinity for FcRn in cynomolgus monkeys (Figure 11).

[0174] [Table 9] The KD(M) value was averaged from 5-6 independent copies.

[0175] Example 6: Sandwich ELISA for cross-reactivity testing Regarding the selected Aβ3pE monoclonal antibody BAMB31, rodent Aβ3pE -40 (Sequence No. 45) and human Aβ1-40 (Sequence No. 41), Aβ1-42 (Sequence No. (No. 48), Aβ11pE-40 (SEQ ID NO: 49) and Aβ11pE-42 (SEQ ID NO: 50) Cross-reactivity with BAMB31+JRF / cAβ was evaluated using synthetic peptides. Using the 40 / 28-HRPO combination, Aβ1-40 (SEQ ID NO: 41), Aβ1 Cross-reactivity with 1pE-40 (SEQ ID NO: 49) and rodent Aβ3pE-40 (SEQ ID NO: 45) We investigated the reactivity and used the BAMB31+JRF / cAβ42 / 26-HRPO combination. This is used to cross-reference 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: Standard substance is diluted with dimethyl sulfoxide (DMSO) (S) at a concentration of 0.1 mg / mL. The peptide was dissolved in PG and stored at -80°C. For use in ELISA, the peptide was converted to P Further diluted to 1 pg / mL with 0.1% casein in BS. 96-well plate (Max isorb ELISA plate (NUNC) in coating buffer, 1.5 μg / Coating overnight at 4°C with monoclonal antibody from BAMB31_1 at a concentration of mL. The next day, the plates were washed and treated with 0.1% casein in PBS at room temperature for 1-4 hours. Blocked. Standard substance was labeled with HRPO-labeled secondary antibody (JRF / cAβ40 / 28-HR Incubated overnight at 4°C with PO or JRF / cAβ42 / 26-HRPO. After incubation overnight, wash the plates and sterilize them with TMB peracid according to the manufacturer's recommendations. The assay was performed using the Biorad EIA substrate kit.

[0177] Results: BAMB31, at concentrations up to 10 ng / mL, showed a reaction with Aβ3pE-40 (sequence). It has selective binding to (number 40) and Aβ3pE-42 (sequence number 51), and is human Aβ1- 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 It was shown that there is no detectable cross-reactivity with 2 (SEQ ID NO: 50) (Figure 4).

[0178] Example 7: Antibody reaction against plaques in transgenic mice and human AD brain tissue Immunohistochemical methods for testing responsiveness The reactivity of antibodies to plaque was measured in formalin-fixed, paraffin-embedded (FFPE) brain tissue. The study was conducted using both cryopreserved brain tissue and cryopreserved brain tissue.

[0179] Materials and methods Formalin-fixed, paraffin-embedded brain: Deparaffinization of sections for immunohistochemical analysis. After fermentation and rehydration, transgenic mouse brains were incubated in formic acid (70% in distilled water) for 10 minutes. Antigen retrieval is performed by incubating the slides, and endogenous peroxidase activity is activated. The properties are broken down with 3% hydrogen peroxide (DAKO (Glostrup, Denmark) S2023). The sections were locked. BAMB246 (huIgG1 chimera), BAMB674 or BAMB675 (Working concentration: Contains background reduction components (DAKO, S3022)) The antibody was incubated with 4 μg / mL of the antibody diluent for 1 hour. A wide area was washed. Afterwards, HRP-labeled anti-human secondary antibody (PI in antibody diluent (DAKO, S0809)) Apply -3000, Vector labs-1 / 500) to the slide for 1 hour, then 3,3-diaminobenzidine (DAB) (DAKO, K3468) was used for colorimetric labeling. The slides were counterstained with hematoxylin, dehydrated, and then treated with Vectormount (H-500). It was permanently fixed using (0, Vector Labs).

[0180] Cryopreserved brain: Human brain samples are rapidly frozen and sliced ​​using a cryostat (20 μm thickness), Stored at -80°C before use. Sections were dried at room temperature, then fixed with formalin, and endogenous phosphate was applied. 3% hydrogen peroxide (DAKO (Glostrup, Denmark) S2) oxidase Blocking in 023), and PBS1x + 0.3% Triton X-100 and Blocking was performed for 1 hour with 10% normal goat serum (DAKO, X0907). Primary Antibody pE3 / 16 (rare antibody with background reduction component (DAKO, S3022)) A solution (2 μg / mL) was applied to the section for 1 hour. After extensive washing, HRP levels were observed. Duplicated anti-mouse secondary antibodies (Envision, DAKO, K4000), followed by The slides were incubated with color pigment DAB labeling (DAKO, K3468). The lides were counterstained with hematoxylin, anhydroused, and fixed in organic medium (Vectamount The subject was fixed using Vector Labs. Imaging was performed at Hamamatsu Nanozoo. mer(Hamamatsu Photonics (Shizuoka, Japan) It was held at ).

[0181] Results: BAMB264 (human IgG1 chimera), BAMB674, and BAMB67 The reactivity of step 5 was demonstrated on FFPE tissue from transgenic mice (Figures 5A-5F). Furthermore, BAMB31_2a(mIgG2a) is found in the parenchyma of cryopreserved AD brain tissue. This showed typical plaque labeling (Figure 6). Substantial plaque labeling was observed in the plaques detected by antibody 4G8. The fraction is also labeled with BAMB31 in cryo-sections of human brain (Figures 5A-5A). 5F). ++

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

[0183] Materials and Methods: Aged tracheostomy cells expressing high concentrations of human Aβ42 and Aβ40 peptides. Sgenic mice (22-23 months old) received 20 mg / kg of mE8c mIgG2a , BAMB31_2a(mIgG2a) or isotype control antibody mIgG2a (treatment group) Each participant (n=5) received a single intraperitoneal (IP) injection. After antibody administration, participants were monitored at an intermediate point (after injection). At 24 hours and 48 hours, use the MICROVETTE® recovery tubes (each 100Z and 300Z, Sarstedt (Numbrecht, Germany) Then, at the time of slaughter (four days after injection), whole blood is collected via the great saphenous vein by orbital puncture. The collected whole blood was incubated at room temperature for 1-2 hours, followed by heating at 10,000 rpm. The serum was separated from the blood clot by centrifugation at 4°C for 10 minutes. The serum antibody titer was determined by allotype. Specific enzyme-linked immunosorbent assay (ELISA) This was determined using Nunc MaxiSorp® flat-bottom plate (T Hermo Scientific (Waltham, MA) at 1.5 μg / mL Mouse monoclonal anti-IgG2a(a) (BD Biosciences (San J Coated overnight at room temperature with ose, CA). mE8c mIgG2a, BAMB3 The antibody standards for 1_2a (mIgG2a) and isotype control mIgG2a were blocked. 1 μg / mL in buffer (PBS buffer + 1% BSA in 0.05% Tween-20) Each sample was prepared individually and further diluted to 0.1 ng / mL in block buffer. After washing (PBS Buffer + 0.05% Tween-20), block sample in block buffer at room temperature for 1 hour. Next, the standard and pre-diluted serum samples were coated with MAXISO. The sample was incubated in an RP(trademark) plate at room temperature for 1 hour. Next, wash the plate and apply Peroxidase-AffiniPure goat anti-mouse Ig Incubate with 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 applied. The color was produced by adding it to the well. By adding 2N H2SO4 to the well... Then, after 2 minutes, the color display stopped, and the EnVision multimode plate reader (Perk The plate was read at 450nm using (in Elmer).

[0184] Results: Serum antibody titers were measured 24 hours, 48 ​​hours, and after intraperitoneal injection of 20 mg / kg of antibody. Measurements were taken 4 days later. The average antibody concentration in serum was measured 24 hours after examination for all antibodies examined. It was equivalent to BAMB31_2a and the isotype control antibody. The decrease occurs gradually over time (decreasing to about 30% by day 4), while on day 4 it reaches about 97%. Regarding the decrease in mE8c, a clearly larger decrease over time was observed. (Figure 7).

[0185] In conclusion, in a mouse model of plaque deposition, BAMB31_2a and mE8c were found to be abnormal. The difference in pharmacokinetic profiles after intraoral injection was shown, with BAMB31 compared to mE8c. A slower clearance was observed after treatment with _2a antibodies.

[0186] Example 9: Study of long-term efficacy in a transgenic mouse model In transgenic mouse models, BAMB31_2a mIgG2a We investigated the effectiveness of reducing amyloid load after long-term treatment, as well as its effect on microbleeding.

[0187] Materials and Methods: PDAPP (V717F) transgenic mice (average at the start of the study) A patient (18.3 months old) was given 30 mg / kg of BAMB31_2a antibody (mIgG2a). Treatment involved intraperitoneal administration once a week for 12 weeks. mIgG2a isotype control antibody injection was administered. A control group that received the treatment was included in the experiment. The animals were euthanized seven days after the final intraperitoneal injection (end of study). (Average age at completion: 21.1 months). Mice were perfused with PBS before collection. Left hemisphere (fractionation) 1: Hippocampus, 2: The remaining part of the brain (excluding the hippocampus, cerebellum, and brainstem), for further biochemical analysis. The right hemisphere was frozen and preserved, while the right hemisphere was fixed overnight in a formalin-based fixative, followed by paraffin The sample was embedded in a film and sliced ​​with a microtome (5 μm).

[0188] To evaluate the effect on amyloid load after long-term treatment, biochemical analysis and immunoassay were performed. Both histochemical and biochemical analyses were performed. For biochemical analysis, Tallprep Lysin was used. Using g Matrix D tubes (MP Bio), ice-cold 5M guanidine-HCl In 1 and 50 mM Tris / HCl extraction buffer (100 mg tissue / mL extraction buffer) The brain was homogenized. After homogenization, the sample was placed in an end-over-end rotating wheel at room temperature for 3 hours. Placed. Before analysis with the MSD sandwich immunoassay, the resulting homogenate was - It was stored at 80°C.

[0189] Synthetic Aβ peptide standard substance is diluted with dimethyl sulfoxide (DMSO) at a concentration of 0.1 mg / mL. Dissolve in Sigma and store at -80°C. For use in MSD immunoassays, Peptides are mixed in 0.5M GuHCl + 5mM Tris-HCl-pH8.0 (PBS). The extract was further diluted with a 10-fold dilution of the extraction buffer in 0.1% casein. The GuHCl extract was then dissolved. Freeze, dilute 1:10 in 0.1% casein in PBS (ice-cold), and incubate at 4°C for 20 minutes. It was centrifuged at 0g. The supernatant was collected for use in the sandwich MSD assay, and 0 .5M GuHCl+5mM Tris-HCl-pH8.0 (0.1% casei in PBS) The sample was further diluted with a 10-fold dilution of the extraction buffer in the solution.

[0190] 96-well sector plate standard product (Meso Scale Discovery (R Rockville (MD) was administered monoclonally in PBS at a concentration of 1.5 μg / mL. The body was used to coat the plate overnight at 4°C. The next day, the plate was washed and coated with 0.1% PBS. The samples were blocked with zein at room temperature for 2 hours. The standard material and the sample were treated with a biotin-labeled secondary antibody. The body was incubated overnight at 4°C. After overnight incubation, the plate was washed. And, a secondary detection reagent (Streptavidin-SULFO-TAG (trademark) labeled) and Both were incubated at room temperature for 2 hours. The plates were washed and 2x Read Buffed. rT was added, and then the plate was read according to the manufacturer's recommendations. 1 / Y 2 Weighted Using a 4-parameter logistic model with a function, the Aβ concentration is calculated using a standard curve. The judgment was made.

[0191] Using the JRF / AβN / 25+4G8 biotin antibody combination, brain homogenates The concentration of Aβ1-x inside was investigated.

[0192] For immunohistochemical analysis, after deparaffinization and rehydration of the sections, formic acid (7 oz in distilled water) was used. Antigen retrieval is performed by incubating the slide in 0% for 10 minutes, and endogenous Sex peroxidase activity is measured using 3% hydrogen peroxide (DAKO, Glostrup, Denmar Blocked by k, S2023). Background reduction component (DAKO, S302 2) In an antibody diluent having a biotinylated 4G8 antibody (Bio) diluted to 1 / 2000 Sections were incubated overnight with Legend (San Diego, CA). After washing the area, streptavidin-HRP (PK6100 Elite, Vect Apply (or labs) to the slide for 30 minutes, followed by 3,3-diaminobenzidine (D Chromogenic labeling was performed using AB) (DAKO, K3468). The slides were treated with hematoxylin. Stained, dehydrated, and then in Vectormount (H-5000, Vector Labs) It was permanently fixed. NanoZoomer slide scanner (Hamamatsu Ph An image (20x) was generated using otonics, and then divided into parts using Matlab / Phaedra. The analysis was conducted. The region-of-interest (ROI) was defined by Franklin and Pa. xinos atlas(Franklin KB,Paxinos G.Mouse brain in stereotaxic coordinates.Waltham Manually delineated according to (Academic Press; 1997), and each ROI Then, the percentage of DAB-labeled regions per total area was calculated.

[0193] Perl staining was performed to evaluate the effect on microbleeds. In short, Paraffin-embedded tissue sections were prepared in acid ferrocyanide solution according to the protocol described below. Processed. The ferric ions (Fe3+) present in the small amount of bleeding combine with ferrocyanides. Together, they result in the formation of a blue pigment called Prussian blue. Deparaffinization and After rehydration, 2% potassium ferrocyanide (Sigma-Aldrich) and 2% ice form The sections were incubated for 30 minutes in a 1 / 1 mixture with hydrochloric acid (Sigma-Aldrich). I did that. After rinsing the slide three times with distilled water, I used nuclear fast re After counterstaining with d(Sigma-Aldrich), distilled water, dehydration, and fixative ( Rinsed with Vectamount (Vector Labs). Imaging was done with Nano The scan was performed using a Zoomer slide scanner (Hamamatsu Photonics). The number of Perl-positive cells near the meninges was counted manually.

[0194] Results: Overall, a small number of patients in the baseline, isotype control, and BAMB31 treatment groups Microbleeding was observed (Figure 8). Biochemical analysis regarding Aβ1-x concentration in the hippocampus , 34% (p<0.0001) of BAMB31_2a compared to isotype control antibody A decrease was observed (Figure 9). In addition, immunohistochemistry using antibody 4G8 showed that the hippocampus and cortex Compared to isotype control antibodies, the results were 23% (p<0.0001) and 37% (p It showed a decrease of <0.001).

[0195] In conclusion, after long-term intraperitoneal injection with BAMB31 in a plaque deposition mouse model... The effectiveness in reducing amyloid load without causing an increase in the occurrence of microbleeds is This demonstrated a favorable efficacy-to-toxicity ratio after treatment with BAMB31 antibodies.

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

[0197] Materials and methods: Three animals per group were given 25 mg / kg of each mAb intravenously (iv). The drug was administered by bolus injection, and serum samples were collected over 5 weeks. At week 6, each mAb was administered. Three additional monkeys were given an intravenous bolus injection of 25 mg / kg of the drug, and on the 7th day after administration and On day 42, brain tissue was collected from each group. In total, for each molecule, on days 7 and 42... Brain samples were collected from three crab-eating macaques.

[0198] Drugs BAMB674 and BAMB675 from in vivo cynomolgus monkey serum and brain tissue samples Material exposure analysis is conducted in Meso Scale Discovery (MSD) Sector I. Individually-purpose electrochemiluminescence with endpoint determination on the Mager S600 The immunoassay (ECLIA) was performed using one assay format for the compound and We obtained four independent methods for applying to each matrix and measuring exposure. The form is explained as follows: It captures mAb compounds and targets anti-human Fc-specific (CH2 dormancy) Detection was performed using mouse mAbs. For brain tissue preparation, rapidly frozen tissue was freeze-pulverized and buffered. Homogenates were prepared by dilution in liquid. The protein concentration in the tissue was then verified. The final protein concentration used in this method was determined using a BCA assay. 5-parameter logistic (automatic estimation) fitting with Y2 standard curve weighting We performed raw data regression using Watson LIMS software.

[0199] Two compartments via intravenous (IV) administration (Admin) into the central compartment. Ment (center (V C ) and organization (VT Pharmacokinetic (PK) models of compartments Using the inter-compartment clearance (Q) and linear clearance (CL), The PK of BAMB674 and BAMB675 was characterized in meat-mollusk monkeys. Figure 10 shows The model outline is shown below.

[0200] Results: The terminal half-life of each antibody was calculated and compared with the data from cynomolgus monkeys in two compartments. The fitting of the ment model is shown in Figure 11. +YTE IgG1 isotype ( BAMB675) is approximately 1.5% higher than the wild-type IgG1 isotype (BAMB674). The half-life increased sixfold. This is because the wild-type IgG1 mAb (BAMB67) is shown in Table 5. 4) The FcRn affinity of the +YTE isotype (BAMB675) increased by approximately 3 times compared to the previous version. It concerns.

[0201] The levels of cerebral lysate across the region and between mAbs were similar on day 7. However, on day 42, only YTE mAb was consistently detected across brain regions and animals. The results are shown in Figure 12. This is due to the increase in exposure to +YTE mAb at later time points. This has been consistent.

[0202] Certain terms are used for clarity when describing the present invention and its various embodiments. However, the present invention is not intended to be limited to such selected terms. Those skilled in the art will recognize that other equivalent components may be used and other methods may be developed without departing from the broader concept of the present invention. All references cited throughout this specification are incorporated by reference as if each were incorporated individually. The invention described in this specification may encompass the following embodiments. [1] a. Sequence numbers 1, 2, 3, 4, 5, and 6, respectively b. Sequence numbers 1, 7, 3, 4, 5, and 6, respectively c. Sequence numbers 1, 7, 3, 8, 5, and 6, respectively d. Sequence numbers 1, 2, 3, 8, 5, and 6, respectively e. Sequence numbers 56, 57, 3, 8, 5, and 6, respectively. f. Sequence numbers 56, 57, 3, 4, 5, and 6, respectively. g. Sequence IDs 56, 58, 3, 4, 5, and 6, respectively h. Sequence numbers 56, 7, 3, 8, 5, and 6, respectively. i. Sequence numbers 1, 57, 3, 8, 5, and 6, respectively j. Sequence numbers 56, 7, 3, 4, 5, and 6, respectively. k. Sequence numbers 1, 57, 3, 4, 5, and 6, respectively. l. Sequence IDs 1, 58, 3, 4, 5, and 6, respectively, or m. An isolated monoclonal antibody or antigen-binding fragment thereof comprising heavy chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, each having the polypeptide sequences of SEQ ID NOs. 56, 2, 3, 4, 5, and 6, wherein the antibody or antigen-binding fragment specifically binds to 3pE Ab, preferably human 3pE Ab. [2] An isolated monoclonal antibody or antigen-binding fragment thereof as described in [1] above, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NOs. 9, 11, 13, 15, 16, 17, 19, 20, or 21, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NOs. 10, 12, 14, 18, 22, 53, or 55. [3] a. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 22 b. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10, c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12 d. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 13 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14, e. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14, f. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 16 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14, g. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 20 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 14, h. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 17 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18, i. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 18, j. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 53, or k. An isolated monoclonal antibody or antigen-binding fragment thereof as described in [1] above, comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 55. [4] The isolated monoclonal antibody or antigen-binding fragment according to any one of the above [1] to [3], wherein the antibody or its antigen-binding fragment is a chimeric antibody. [5] The isolated monoclonal antibody or antigen-binding fragment according to any one of the above [1] to [4], wherein the antibody or its antigen-binding fragment is human or humanized. [6] a. Heavy chain amino acid sequence containing SEQ ID NO: 37 and light chain amino acid sequence containing SEQ ID NO: 38 b. Heavy chain amino acid sequence containing SEQ ID NO: 39 and light chain amino acid sequence containing SEQ ID NO: 38 c. A heavy chain amino acid sequence containing SEQ ID NO: 37 and a light chain amino acid sequence containing SEQ ID NO: 52, or d. Isolated monoclonal antibodies containing a heavy chain amino acid sequence including SEQ ID NO: 39 and a light chain amino acid sequence including SEQ ID NO: 54. [7] An isolated nucleic acid encoding a monoclonal antibody or its antigen-binding fragment as described in any one of the above items [1] to [6]. [8] A vector containing the isolated nucleic acid described above [7]. [9] A host cell containing the vector described above [8].

[10] A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of the above items [1] to [6], and a pharmaceutically acceptable carrier.

[11] A method for treating a condition associated with the formation of plaques containing β-amyloid protein in a subject requiring such treatment, the method comprising administering a monoclonal antibody or an antigen-binding fragment thereof as described in any one of the above items [1] to [6], or a pharmaceutical composition as described in

[10] , to the subject requiring treatment.

[12] The method described above

[11] , wherein the condition is Alzheimer's disease.

[13] The method according to

[11] above, wherein the condition is selected from the group consisting of dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and hereditary cerebral hemorrhage with Dutch type amyloidosis (HCHWA-D).

[14] A method for reducing plaques associated with Alzheimer's disease in a subject requiring such reduction, the method comprising administering a monoclonal antibody or an antigen-binding fragment thereof as described in any one of the above items [1] to [6], or a pharmaceutical composition as described in

[10] , to the subject requiring reduction.

[15] A method for preventing the seeding activity of 3pE Aβ in a subject requiring such activity, the method comprising administering to the subject a monoclonal antibody or antigen-binding fragment thereof as described in any one of the above items [1] to [6], or a pharmaceutical composition as described in

[10] .

[16] A method for producing a monoclonal antibody or an antigen-binding fragment thereof as described in any one of the above items [1] to [6], comprising: culturing cells containing nucleic acids encoding the monoclonal antibody or the antigen-binding fragment under conditions for producing the monoclonal antibody or the antigen-binding fragment; and recovering the antibody or the antigen-binding fragment thereof.

[17] A method for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of the above items [1] to [6], the method comprising combining the monoclonal antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

Claims

1. A method for detecting an amyloid-β peptide (3pE Aβ) or a fragment thereof having a pyroglutamate as the third residue in a biological sample, wherein the method is: (i) Contacting the biological sample with a monoclonal antibody or antigen-binding fragment thereof that specifically binds to 3pE Aβ or a fragment thereof, and (ii) To determine whether an immune complex is formed between the monoclonal antibody or its antigen-binding fragment and the 3pE Aβ or its fragment. Includes, Here, the monoclonal antibody or its antigen-binding fragment is a. Sequence numbers 1, 2, 3, 4, 5, and 6, respectively b. Sequence IDs 1, 7, 3, 8, 5, and 6, respectively c. Sequence IDs 56, 57, 3, 8, 5, and 6, respectively, or d. Each comprising heavy chain complementarity determination region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determination region 1 (LCDR1), LCDR2, and LCDR3, each having the polypeptide sequence of sequence numbers 56, 58, 3, 4, 5, and 6, method.

2. The method according to claim 1, wherein the monoclonal antibody or the antigen-binding fragment thereof includes a heavy chain variable region having a polypeptide sequence identical to at least 95% of SEQ ID NO: 9 or 21, or a light chain variable region having a polypeptide sequence identical to at least 95% of SEQ ID NO: 10, 12, 22, 53, or 55.

3. The monoclonal antibody or its antigen-binding fragment is a. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 22 b. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10, c. A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 53, or d. Heavy chain variable region having the polypeptide sequence of SEQ ID NO: 21 and light chain variable region having the polypeptide sequence of SEQ ID NO: 55 The method according to claim 1 or 2, including the method described in claim 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the monoclonal antibody or its antigen-binding fragment is a chimeric antibody.

5. The method according to any one of claims 1 to 4, wherein the monoclonal antibody or its antigen-binding fragment is humanized.

6. The method according to any one of claims 1 to 5, wherein the monoclonal antibody or its antigen-binding fragment specifically binds to human 3pE Aβ.

7. The monoclonal antibody is a. Heavy chain amino acid sequence containing SEQ ID NO: 37 and light chain amino acid sequence containing SEQ ID NO: 38 b. A heavy chain amino acid sequence containing SEQ ID NO: 39 and a light chain amino acid sequence containing SEQ ID NO: 38, c. A heavy chain amino acid sequence containing SEQ ID NO: 37 and a light chain amino acid sequence containing SEQ ID NO: 52, or d. Heavy chain amino acid sequence containing SEQ ID NO: 39 and light chain amino acid sequence containing SEQ ID NO: 54 The method according to any one of claims 1 to 6, including the method described in any one of claims 1 to 6.

8. The method according to any one of claims 1 to 7, wherein the biological sample is a tissue sample.

9. The method according to any one of claims 1 to 7, wherein the biological sample is a body fluid sample.

10. The method according to claim 9, wherein the body fluid sample is selected from cerebrospinal fluid samples, blood samples, plasma samples, serum samples, and urine samples.

11. The method according to any one of claims 1 to 10, wherein the biological sample is derived from an object having a state associated with the formation of plaques containing β-amyloid protein.

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

13. The method according to claim 11, wherein the condition is selected from the group consisting of dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and hereditary cerebral hemorrhage with Dutch type amyloidosis (HCHWA-D).

14. The monoclonal antibody or its antigen-binding fragment comprises a detectable label, The method according to any one of claims 1 to 13, further comprising detecting the detectable sign.

15. The method according to claim 14, wherein the detectable label is selected from radioisotopes, enzymes, fluorescent substances and luminescent substances.

16. The method according to any one of claims 1 to 15, wherein the monoclonal antibody or its antigen-binding fragment is immobilized on a solid matrix.

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