Antibodies to pyroglutamate amyloid-beta and uses thereof

Abstract

The present invention relates to antibodies to pyroglutamate amyloid-beta and uses thereof. In particular, the present invention provides antibodies, or antigen-binding fragments thereof, that bind to 3pE A [beta], as well as methods of making and using the antibodies, or antigen-binding fragments thereof, including uses in formulations, administration, and kits. The disclosed antibodies, antigen binding fragments thereof, and methods are useful in the diagnosis, prognosis, and treatment of Alzheimer's disease or other beta-amyloid related diseases.

Description

[0001] This application is a divisional application of the patent application with the original application date of March 25, 2020, application number 202080039618.2 (international application number PCT / EP2020 / 058395), and invention name “Antibodies against pyroglutamate amyloid protein-β and their uses”. Technical Field

[0002] The present invention relates to the field of antibodies against amyloid-β (Aβ) peptide and therapeutic methods using the same. In particular, the antibodies can be used to identify and treat amyloid-related diseases.

[0003] Reference sequence listing submitted electronically

[0004] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name of "JAB7013USPSP Sequence Listing" and a creation date of March 11, 2019, and is 76 kb in size. This sequence listing, submitted via EFS-Web, is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0005] Alzheimer's disease (AD) is a degenerative brain disorder characterized by a progressive loss of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to extreme mental decline and eventual death. Alzheimer's disease is a common cause of progressive mental decline (dementia) in the elderly. Alzheimer's disease has been found worldwide and represents a major public health problem. It is estimated that only in the United States, the disease currently affects more than about 5 million people. It is currently incurable, and any treatment is not effective in preventing AD or reversing its symptoms or process.

[0006] The brains of individuals with AD display characteristic lesions called amyloid plaques, amyloid angiopathy (deposition of amyloid in blood vessels), and neurofibrillary tangles. These lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in large numbers in several areas of the brain that are important for memory and cognitive function. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with trisomy 21 (Down syndrome), diffuse Lewy body disease, and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D).

[0007] The main components of amyloid plaques are various amyloid-β (Aβ) peptides, which are produced by the cleavage of the β-amyloid precursor protein (APP). It is hypothesized that the deposition of Aβ peptides in the brain is an early and necessary step in the disease cascade leading to AD. The identification of mutations in the amyloid precursor protein and presenilin genes that lead to altered Aβ production and early onset of AD in families provides strong evidence that altered amyloid metabolism is a central event in the pathogenic process that underlies the disease.

[0008] Amyloid-β peptide (3pE Aβ) with pyroglutamate on the third residue is the main substance deposited in the brain of AD patients. 3pE Aβ is present in almost all diffuse and mature plaques in AD, is metabolically stable and can play a role in both plaque inoculation and stabilization (Cynis et al., Molecular Neurodegeneration, 2016; 11: 48). Detectable amounts of 3pE Aβ in CSF or plasma have not yet been reported, indicating that the target peptide is pathologically specific (DeMattos et al., Neuron, 2012; 76: 1-13). Antibodies that selectively bind to 3pE Aβ can be used for immunotherapy. Summary of the Invention

[0009] As embodied and fully described, the present invention relates to antibodies and antigen-binding fragments thereof that bind to amyloid-β with pyroglutamate at the third residue (3pE Aβ), methods for producing antibodies or antigen-binding fragments thereof that bind to 3pE Aβ, assays using such antibodies or antigen-binding fragments thereof, and the use of the antibodies or antigen-binding fragments thereof of the invention in the manufacture of a medicament for treating Alzheimer's disease and other β-amyloid-related diseases, delaying their onset, or reversing at least one pathology or symptom thereof. The antibodies of the invention preferentially bind to Aβ peptides containing 3pE compared to Aβ peptides that do not contain 3pE.

[0010] Specifically, described herein are isolated monoclonal antibodies or antigen-binding fragments thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the following polypeptide sequences:

[0011] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0012] b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively;

[0013] c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively;

[0014] d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively;

[0015] e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively;

[0016] f. SEQ ID NOs: 56, 57, 3, 4, 5 and 6, respectively;

[0017] g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively;

[0018] h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively;

[0019] i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively;

[0020] j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively;

[0021] k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively;

[0022] l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or

[0023] m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively;

[0024] The antibody or antigen-binding fragment thereof specifically binds to 3pE Aβ, preferably human 3pE Aβ.

[0025] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment comprises a heavy chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 9, 11, 13, 15, 16, 17, 19, 20, or 21, or a light chain variable region having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 10, 12, 14, 18, 22, 53, or 55.

[0026] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0027] a. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0028] a light chain variable region of the polypeptide sequence of 22;

[0029] b. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 9 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0030] 10;

[0031] c. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 11 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0032] A light chain variable region of the polypeptide sequence of 12;

[0033] d. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 13 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0034] A light chain variable region of the polypeptide sequence of 14;

[0035] e. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 15 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0036] A light chain variable region of the polypeptide sequence of 14;

[0037] f. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 16 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0038] A light chain variable region of the polypeptide sequence of 14;

[0039] g. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 20 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0040] A light chain variable region of the polypeptide sequence of 14;

[0041] h. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 17 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0042] A light chain variable region of the polypeptide sequence of 18;

[0043] i. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 19 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0044] A light chain variable region of the polypeptide sequence of 18;

[0045] j. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0046] 53; or

[0047] k. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0048] The light chain variable region of the polypeptide sequence of 55.

[0049] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is chimeric.In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is human or humanized.

[0050] In certain embodiments, the isolated monoclonal antibody comprises:

[0051] a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0052] b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0053] c. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 52; or

[0054] d. A heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 54.

[0055] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fv, F(ab'), F(ab')2, and scFv. The antibody or antigen-binding fragment thereof selectively binds to 3pE Aβ peptide (e.g., Aβ3pE-40 and Aβ3pE-42) with little or no cross-reactivity to other Aβ peptides or β-amyloid precursor protein (APP).

[0056] Also provided are isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof disclosed herein.

[0057] Also provided are vectors comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.

[0058] Also provided are host cells comprising a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention. Also provided are hybridomas producing the isolated monoclonal antibody or antigen-binding fragment thereof of the invention.

[0059] In certain embodiments, a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier is provided.

[0060] Also provided are methods for treating a condition associated with the formation of plaques containing beta-amyloid in a subject in need thereof. The method comprises administering a monoclonal antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention to a subject in need thereof. In certain embodiments, the condition is Alzheimer's disease. In certain embodiments, 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 amyloidosis (HCHWA-D).

[0061] Also provided is a method for reducing plaques associated with Alzheimer's disease in a subject in need thereof, the method comprising administering to a subject in need thereof a monoclonal antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention.

[0062] Also provided is a method for preventing the vaccination activity of 3pE Aβ in a subject in need thereof. The method comprises administering to a subject in need thereof a monoclonal antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention.

[0063] Also provided is a method of producing the monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions that produce the monoclonal antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof.

[0064] Also provided is a method for producing the pharmaceutical composition of the present invention, which comprises combining the monoclonal antibody or antigen-binding fragment thereof of the present invention with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0065] One embodiment includes kits and devices comprising the above-described antibodies or antigen-binding fragments thereof.

[0066] Other objects, features and advantages of the present invention will become apparent to those skilled in the art through detailed consideration of the following preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Figure 5 is a sensorgram of surface plasmon resonance label-free detection of the affinity binding interaction of BAMB31_2a (mIgG2a) with human Aβ (3pE-40) peptide (single cycle kinetics). The grey trace represents double reference subtracted data, while the black trace represents the fitted value.

[0068] Figure 2 Figure 5 is a sensorgram of surface plasmon resonance label-free detection of the affinity binding interaction of mE8c mIgG2a with human Aβ (3pE-40) peptide (single cycle kinetics). The grey trace represents double reference subtracted data, while the black trace represents the fitted value.

[0069] Figures 3A to 3I Shown are the reactivity of BAMB674 and BAMB675 to plaques in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue analyzed by immunohistochemistry. Results are shown for a primary antibody concentration of 0.05 μg / mL. Arrows indicate areas of plaque labeling by BAMB674 and BAMB675. (A) BAMB674; (B) BAMB675; (C) Antibody I; (D) Antibody II; (E) B12L; (F) CI-C7; (G) hE8L; (H) R17L; (I) R17.

[0070] Figures 4A to 4B Shown are graphs demonstrating the selectivity of BAMB31_1 as shown by detection of synthetic human Aβ peptides in a sandwich ELISA. (A) Aβ1-40 (B) AβpE11-40.

[0071] Figures 5A to 5F Shown are the reactivity of (A to B) BAMB246 (huIgG1 chimera), (C to D) BAMB674, and (E to F) BAMB675 to plaques analyzed by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue. Insets show whole-mount stained brain sections and magnified areas.

[0072] 6A to 6D Shown are reactivity to plaques analyzed by immunohistochemistry in cryopreserved AD brain tissue with (A, C) 4G8 and (B, D) BAMB31_2a (mIgG2a) at two different magnifications.

[0073] Figure 7 Shown is a graph demonstrating serum antibody concentrations at different time points following a single 20 mg / kg intraperitoneal (ip) dose in transgenic mice.

[0074] Figure 8 Shown is a graph demonstrating microhemorrhages in PDAPP mice following long-term treatment with isotype control and BAMB31_2a (mIgG2a) antibodies by evaluating the number of Perls-positive cells.

[0075] Figure 9 A graph demonstrating amyloid burden in the hippocampus of PDAPP mice following chronic treatment with isotype control and BAMB31_2a (mIgG2a) antibodies, as measured by an immunoassay detecting Aβ1-x. Grey values represent data points below the detection limit of the assay.

[0076] Figure 10Schematic diagram of the two-compartment model for PK characterization of BAMB674 and BAMB675 monkeys is shown.

[0077] Figure 11 Figures demonstrating PK data and observational data for BAMB674 and BAMB675 are shown. Serum levels of BAM31 HFA mAb (WT serum) and +YTE IgG1 (YTE serum) isotypes as wild-type IgG1 following intravenous (iv) bolus administration of 25 mg / kg in cynomolgus monkeys. Anti-Aβ3pE antibody (3pE-AB) μg / ml concentrations are plotted on a logarithmic scale on the Y-axis versus time in days on the X-axis. The calculated half-life (t1 / 2) of each mAb is shown in the inset text.

[0078] Figure 12 Shown is a graph illustrating brain concentrations observed for BAMB674 and BAMB675. Brain lysate levels on day 7 and day 42 of BAMB31 HFA mAb as wild-type IgG1 (WT brain) and +YTE IgG1 (YTE brain) isotypes following iv bolus administration of 25 mg / kg in cynomolgus monkeys. Anti-Aβ3pE antibody (3pE-AB) μg / ml concentrations are shown on a logarithmic scale on the Y-axis versus time in days on the X-axis. DETAILED DESCRIPTION

[0079] Various publications, articles, and patents are cited or described in the Background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like included in this specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0080] Should be understood that the present invention is not limited to specific method, reagent, compound, composition, or biological system, and this method, reagent, compound, composition, or biological system can change.In addition should be understood that the terminology used herein is just for the purpose of describing specific embodiment, is not intended to limit.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification.

[0082] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0083] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0084] As used herein, the terms "comprises," "including," "having," or "containing," or any other variations thereof, should be understood to mean the inclusion of the stated integer or groups of integers but not the exclusion of any other integer or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, condition A or B is satisfied by any of: A being true (or present) and B being false (or absent), A being false (or absent) and B being true (or present), and both A and B being true (or present).

[0085] As used herein, the connection term "and / or" between multiple listed elements is understood to include both individual options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0086] As used herein, the term "consisting of" used throughout the specification and claims is intended to include any recited integer or group of integers, but does not imply that additional integers or groups of integers may be added to a specified method, structure, or composition.

[0087] As used herein, the term "consisting essentially of" as used throughout the specification and claims means including any recited integer or group of integers, and optionally including any recited integer or group of integers that does not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2111.03.

[0088] Antibody

[0089] The present invention provides antibodies or antigen-binding fragments thereof that bind to 3pEβ peptide, in particular antibodies or antigen-binding fragments thereof that preferentially bind to Aβ peptides that do not contain 3pE. The present invention also provides methods for preparing antibodies or antigen-binding fragments thereof that bind to 3pE Aβ peptide, and methods for preparing hybridomas that produce antibodies or antigen-binding fragments thereof that bind to 3pE Aβ peptide. The present invention also includes methods for treating Alzheimer's disease and other β-amyloid-related diseases in individuals, methods for clearing plaques associated with Alzheimer's disease or other β-amyloid-related diseases, and methods for preventing plaque seeding activity of 3pE Aβ. The present invention also provides kits and devices comprising antibodies or antigen-binding fragments thereof for use in the methods described herein.

[0090] According to a specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequence:

[0091] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0092] b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively;

[0093] c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively;

[0094] d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively;

[0095] e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively;

[0096] f. SEQ ID NOs: 56, 57, 3, 4, 5 and 6, respectively;

[0097] g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively;

[0098] h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively;

[0099] i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively;

[0100] j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively;

[0101] k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively;

[0102] l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or

[0103] m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively;

[0104] The antibody or antigen-binding fragment thereof specifically binds to 3pE Aβ, preferably human 3pE Aβ.

[0105] According to another specific aspect, the present invention is directed to an isolated monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 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 NO: 10, 12, 14, 18, 22, 53 or 55.

[0106] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising:

[0107] 1. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0108] a light chain variable region of the polypeptide sequence of 22;

[0109] m. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 9 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0110] 10;

[0111] n. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 11 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0112] A light chain variable region of the polypeptide sequence of 12;

[0113] o. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 13 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0114] A light chain variable region of the polypeptide sequence of 14;

[0115] p. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 15 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0116] A light chain variable region of the polypeptide sequence of 14;

[0117] q. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 16 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0118] A light chain variable region of the polypeptide sequence of 14;

[0119] r. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 20 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0120] A light chain variable region of the polypeptide sequence of 14;

[0121] s. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 17 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0122] A light chain variable region of the polypeptide sequence of 18;

[0123] t. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 19 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0124] A light chain variable region of the polypeptide sequence of 18;

[0125] u. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0126] 53; or

[0127] v. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0128] The light chain variable region of the polypeptide sequence of 55.

[0129] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 2, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 58, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively, or SEQ ID NO: 1, 58, 3, 4, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 21 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 22 or 53 or 55. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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 or 53 or 55.

[0130] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 2, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 58, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively, or SEQ ID NO: 1, 58, 3, 4, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 20 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0131] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 1, 57, 3, 8, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 19 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 18. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0132] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 1, 57, 3, 8, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 17 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 18. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0133] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 1, 57, 3, 8, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 16 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0134] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 1, 57, 3, 8, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 15 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0135] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively, or SEQ ID NO: 56, 7, 3, 4, 5 and 6, respectively, or SEQ ID NO: 1, 57, 3, 8, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 13 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0136] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 7, 3, 8, 5 and 6, respectively, or SEQ ID NO: 56, 57, 3, 8, 5 and 6, respectively, or SEQ ID NO: 56, 7, 3, 8, 5 and 6, respectively, or SEQ ID NO: 1, 57, 3, 8, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 11 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 12. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0137] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a polypeptide sequence of SEQ ID NO: 1, 7, 3, 8, 5 and 6, respectively, or SEQ ID NO: 56, 57, 3, 8, 5 and 6, respectively, or SEQ ID NO: 56, 7, 3, 8, 5 and 6, respectively, or SEQ ID NO: 1, 57, 3, 8, 5 and 6, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 9 and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 10. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises 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.

[0138] In another specific aspect, the isolated monoclonal antibody comprises:

[0139] a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0140] b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0141] c. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 52; or

[0142] d. A heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 55.

[0143] According to another specific aspect, the present invention relates to the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof is chimeric.

[0144] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof is human or humanized.

[0145] According to another specific aspect, the present invention relates to an antigen-binding fragment, wherein the antigen-binding fragment is selected from the group consisting of Fv, F(ab'), F(ab')2, and scFv. The antibody or antigen-binding fragment thereof selectively binds to 3pE Aβ peptide (e.g., Aβ3pE-40 and Aβ3pE-42) with little or no cross-reactivity with other Aβ peptides or β-amyloid precursor protein (APP).

[0146] In another general aspect, the present invention relates to isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof of the present invention. Those skilled in the art will appreciate that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will appreciate that the nucleic acid sequence encoding a monoclonal antibody or antigen-binding fragment thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0147] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody of the present invention or its antigen-binding fragment. According to the present disclosure, any vector known to those skilled in the art, such as a plasmid, a cosmid, a phage vector or a viral vector, can be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element of the conventional function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selective marker and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A variety of expression vectors capable of delivering nucleic acid to cells are known in the art and can be used herein to produce antibodies or their antigen-binding fragments in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present invention.

[0148] In another general aspect, the present invention relates to a host cell comprising a nucleic acid encoding the separation of a monoclonal antibody of the present invention or its Fab. In view of the present disclosure, any host cell known to those skilled in the art can be used for recombinantly expressing the antibody of the present invention or its Fab. In some embodiments, the host cell is an Escherichia coli TG1 or BL21 cell (for expressing, for example, scFv or Fab antibody), a CHO-DG44 or CHO-K1 cell or a HEK293 cell (for expressing, for example, a full-length IgG antibody). According to a specific embodiment, the recombinant expression vector is transformed into a host cell by conventional methods such as chemical transfection, heat shock or electroporation, wherein the recombinant expression vector is stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0149] In another general aspect, the present invention relates to a method for producing a monoclonal antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing a cell comprising a nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof under conditions that produce the monoclonal antibody or antigen-binding fragment thereof of the present invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cell and purified according to conventional techniques known in the art and as described herein.

[0150] The present invention provides isolated antibodies or antigen-binding fragments thereof that bind to 3pE Aβ. The term "antibody" herein refers to an immunoglobulin capable of binding to an antigen or a portion thereof, in particular an immunoglobulin capable of specifically binding to 3pE Aβ. Antibody binding to an antigen can be measured by methods known to those skilled in the art, for example, using BIAcore TM An antibody or antigen-binding antibody fragment is said to specifically bind an antigen when the dissociation constant is less than or equal to 1 μM, preferably less than or equal to 100 nM, and most preferably less than or equal to 10 nM.

[0151] The antigen-binding fragment of an antibody refers to a fragment of an antibody that can bind to the antigen to which the intact antibody binds and competes with the intact antibody for antigen binding. The antigen-binding fragment comprises a portion of the intact antibody that allows antigen binding (i.e., the variable region of the intact antibody). Antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), single-chain antibody molecules (e.g., scFV), diabodies, minibodies, nanobodies, linear antibodies, single-domain antibodies (sdab), camel-humped single-domain antibodies, multispecific antibodies formed by antibody fragments, and any other antibody fragments that bind to an antigen but do not contain the structure of an intact antibody.

[0152] Antibodies are composed of two heavy chains and two light chains. Each heavy chain has a variable domain or region (V H ), followed by a constant domain or region (C H 1), hinge region, and two other constant domains or regions (C H 2 and C H 3). Each light chain has a variable domain or region (V L ) and a constant domain or region (C LThe variable domains or regions of the heavy and light chains form the paratope (a lock-like structure) of the antibody, which is specific for a particular epitope (similar to a key), allowing the paratope and epitope to bind together in a precise manner. Within the variable domain, the variable loops of the beta chains (three each on the light and heavy chains) are responsible for binding to the antigen. These loops are called complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3).

[0153] CDR is defined as the complementary determining region of antibody. These are the hypervariable regions of the antibody heavy chain and light chain that are primarily responsible for being bonded to the antigen. There are three CDRs (CDR1, CDR2 and CDR3) in each of the heavy chain and light chain variable regions. The light chain variable complementary determining region is alternatively referred to as LCDR1, LCDR2 and LCDR3, and the heavy chain variable complementary determining region is alternatively referred to as HCDR1, HCDR2 and HCDR3. The CDR of antibody can be defined in a variety of ways. For example, the CDR in the variable region can be identified according to the definition and / or conformational definition of Kabat, Chothia, IMGT or any CDR determination method well known in the art. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat (Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, NIH, Washington DC), structural loop structures originally defined by Chothia (Chothia et al., Nature 342:877-883 (1989)), or by the unique numbering system of IMGT (Lefranc, The Immunologist 7:132-136 (1999); Lefranc et al., Nucleic Acids Res. 27:209-212 (1999); Scavner et al., Exp. Clin. Immunogenet. 16:234-240 (1999); Lefranc et al., Nucleic Acids Res. 43:D413-422 (2015)).

[0154] When used in the context of an antibody, "isolated" means altered "by the hand of man" from any natural state; that is, if it occurs in nature, it has been altered or removed from its original environment, or both. For example, a naturally occurring antibody that occurs naturally in a living animal in its natural state is not "isolated," but the same antibody separated from the coexisting materials of its natural state is "isolated," as the term is used herein. For example, an "isolated antibody" may refer to an antibody that is substantially free of other antibodies with different antigenic specificities (i.e., an isolated antibody that specifically binds to 3pEAβ is substantially free of antibodies that do not bind to 3pEAβ). An antibody may be present in a composition, such as an immunoassay reagent, that is not a naturally occurring composition and still be an isolated antibody within the meaning of the term (as used herein).

[0155] The method for producing antibodies comprises inoculating the host with the immunogen of expectation.Suitable hosts include but are not limited to mice, rats, hamsters, guinea pigs, rabbits, chickens, donkeys, horses, monkeys, chimpanzees, orangutans, gorillas, people and any species that can establish a mature immune response.Immunoprogram is well established in this area and is described in many papers and publications, including "The Immunoassay Handbook" (2nd edition, David Wild edits, Nature Publishing Group, 2000).

[0156] Preferably, the immunogen comprising the features of the present invention is administered to a host subject, such as an animal or a human, in combination with an adjuvant. Suitable adjuvants include, but are not limited to, Freund's adjuvant, powdered aluminum hydroxide (alum), aluminum hydroxide together with Bordetella pertussis, and monophosphoryl lipid A-synthetic trehalose distarch mycolate (MPL-TDM).

[0157] Typically, the immunogen or a combination of an immunogen and an adjuvant is injected into a mammalian host by one or more subcutaneous or intraperitoneal injections. Preferably, the immunization protocol is carried out over at least one week, and more preferably over two or more weeks. The polyclonal antibodies produced in this manner can be isolated and purified using methods well known in the art.

[0158] Monoclonal antibodies can be produced by the well-established hybridoma method of Kohler and Milstein, e.g., Nature 256:495-497 (1975). The hybridoma method generally involves isolating lymphocytes from a host or a host, harvesting the lymphocytes that secrete or have the potential to secrete the monoclonal antibody, fusing the lymphocytes to immortalized cells, and selecting cells that secrete the desired monoclonal antibody.

[0159] The host can be immunized to induce lymphocytes that produce or are capable of producing antibodies specific for the immunization. Alternatively, the lymphocytes can be immunized in vitro. If human cells are desired, peripheral blood lymphocytes can be used, but spleen cells or lymphocytes from other mammalian sources are preferred.

[0160] Lymphocytes can be fused with immortalized cell lines to form hybridoma cells, which is a process that can be promoted by using a fusing agent, such as polyethylene glycol. By way of example, it is possible to use mutant rodents, cattle or human myeloma cells that are immortalized by transformation. With respect to unfused immortalized cells, it is preferred that substantially pure fusion tumor cell groups be generated. Therefore, after fusion, cells can be grown in a suitable culture medium that suppresses the growth or survival of unfused immortalized cells, such as by using mutant myeloma cells that lack hypoxanthine guanine phosphoribosyltransferase (HGPRT). In such cases, hypoxanthine, aminopterin and thymidine can be added to culture medium (HAT culture medium) to prevent the growth of HGPRT-deficient cells that allow hybridoma growth.

[0161] Preferably, immortalized cells that fuse efficiently can be isolated from a mixed population by selection in a medium such as HAT and support stable and high-level expression of the antibody after fusion. Preferred immortalized cell lines include myeloma cell lines purchased from the American Type Culture Collection (Manassas, VA).

[0162] One aspect of the present invention is a method for producing a hybridoma cell line capable of producing a monoclonal antibody that binds to an amyloid beta peptide. Such methods are generally known to those skilled in the art and generally comprise: (i) selecting a host for antibody production; (ii) inoculating the host with a desired immunogen; (iii) fusing a cell line of the inoculated host with continuously dividing cells to generate a fused cell capable of producing a monoclonal antibody that binds to the immunogen; and (iv) cloning the fused cell to obtain a hybridoma cell line.

[0163] The methods of the present invention include methods for generating hybridoma cell lines capable of producing monoclonal antibodies that bind to the 3pE Aβ peptide. Hybridomas can be prepared by immunizing animals (such as Balb / c mice) from which hybridomas can be produced with an initial intraperitoneal injection of the desired immunogen (such as Aβ peptide with pyroglutamic acid in Freund's adjuvant), followed by additional injections, for example, every one to two weeks. Subsequent fusion of isolated spleens can be performed using any technique generally known to those of ordinary skill in the art, preferably using SP2 / 0 cells, using a modified procedure of Kohler and Milstein (Eur. J. Immunol., 1976; 6: 292-295). Hybridomas can be screened to determine which hybridomas produce antibodies specific for the 3pE Aβ peptide. Screening can be performed in standard assays such as ELISA or RIA assays. One aspect of the present invention is a method for generating a hybridoma cell line that produces the monoclonal antibody BAMB31_1 or a humanized form thereof.

[0164] Monoclonal antibodies can also be produced by recombinant methods known in the art, for example, as described in U.S. Patent No. 4,166,452. DNA encoding the monoclonal antibodies can be isolated and sequenced using conventional procedures, for example, using oligonucleotide probes that bind specifically to murine antibody heavy and light chain genes, preferably probe DNA isolated from a monoclonal antibody hybridoma cell line that secretes Aβ-specific antibodies with pyroglutamate.

[0165] Antibody fragments containing a specific binding site for the amyloid beta peptide can also be produced. Such fragments include, but are not limited to, F(ab')2 fragments, which can be produced by pepsin digestion of antibody molecules; and Fab fragments, which can be generated by reducing the disulfide bonds of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science 256:1270-1281 (1989)). Fab, Fv, and ScFv antibody fragments can all be expressed and secreted in E. coli, allowing the production of large quantities of these fragments. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., BioTechnology 10:163-167 (1992)). Other techniques for producing antibody fragments are known to those skilled in the art. Single-chain Fv fragments (scFv) are also contemplated (see, e.g., U.S. Patent Nos. 5,761,894 and 5,587,458). Fv and sFv fragments are unique in having complete combining sites that lack constant regions; therefore, they may show reduced nonspecific binding. The antibody fragment may also be a "linear antibody," as described, e.g., in U.S. Patent No. 5,642,870.

[0166] Therefore, an object of the present invention is to provide an isolated monoclonal antibody expressed by the above hybridoma cells, which can specifically recognize 3pE Aβ. The isolated monoclonal antibody can be expressed by hybridoma cells or recombinantly.

[0167] Preferably, the antibodies or antigen-binding fragments thereof of the present invention selectively bind to 3pE Aβ, with little or no cross-reactivity with other Aβs that do not have 3pE or β-amyloid precursor protein (APP). Specifically, the antibodies or antigen-binding fragments thereof of the present invention selectively bind to Aβ3pE-40 (SEQ ID NO: 40 or SEQ ID NO: 45) and Aβ3pE-42 (SEQ ID NO: 51) peptides, with little or no cross-reactivity with other Aβ peptides that do not contain 3pE or APP.

[0168] The amino acid sequences of the antibodies of the invention are provided in Table 1. The CDRs of the heavy and light chain variable regions as defined by Kabat, Chothia and IMGT are shown in separate sequences.

[0169] Table 1: 3pEAβ monoclonal antibody sequences

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-3pE Aβ antibodies and polynucleotides encoding them, 3pE Aβ polypeptides and 3pE Aβ polynucleotides encoding them) refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0184] For sequence comparison, one sequence is typically used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified (if necessary), and program parameters for the sequence algorithm are specified. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence based on the specified program parameters.

[0185] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by using the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48: 443 (1970), by searching for similarity of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988). 85:2444 (1988)), by such algorithms (GAP, BESTFIT, FASTA, and TFASTA, computerized implementations in the Wisconsin Genetics Software Package, Genetics Computing Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Laboratory Protocols, a joint venture of Greene Publishing Associates and John Wiley & Sons (1995 supplement) (Ausubel)).

[0186] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Res. Vol. 25: pp. 3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive-valued threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0187] For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hit in each direction stops when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains by default. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0188] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, Vol. 90, pp. 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0189] Another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0190] In vitro methods

[0191] It should be understood that all forms of immunoassays employing antibodies or antigen-binding fragments thereof are contemplated for use according to the presently preferred embodiments, including assays in which the antibodies or antigen-binding fragments thereof are bound to a solid phase and assays in which the antibodies are in a liquid medium. Immunoassay methods that can be used to detect analytes using antibodies embodying features of the present invention include, but are not limited to, competitive (reagent-limited) assays in which a labeled analyte (analyte analog) in the sample and the analyte competes for the antibody, as well as single-site immunoassays in which the antibody is labeled; etc.

[0192] The antibodies or antigen-binding fragments thereof according to the present invention can be used in conventional immunological techniques for detecting Aβ3pE wherever it may be present, including biological samples for monitoring beta-amyloid-related diseases and conditioned medium from cell culture for monitoring intracellular processing of APP. Suitable immunological techniques are well known to those skilled in the art and include, for example, ELISA, Western blot analysis, competitive or sandwich immunoassays, and the like, and as is also well known, all rely on the formation of antigen-antibody immune complexes, wherein for the purpose of the assay, the antibody or antigen-binding fragment thereof can be detectably labeled with, for example, a radioactive, enzymatic, luminescent or fluorescent label, or can be immobilized on an insoluble support. Therefore, the object of the present invention is to provide an immunoassay for determining or detecting Aβ3pE or a fragment thereof in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof directed against Aβ3pE or a fragment thereof according to the present invention, and determining whether an immune complex is formed between the antibody or antigen-binding fragment thereof and Aβ3pE or a fragment thereof. These methods can be performed on tissue samples or body fluid samples and generally include obtaining a sample from the body of a subject; contacting the sample with an imaging-effective amount of a detectably labeled antibody or antigen-binding fragment thereof according to the present invention; and detecting the label to determine the presence of Aβ3pE or a fragment thereof in the sample. There is no particular limitation on the measurement method using the antibody or antigen-binding fragment thereof of the present invention. Any measurement method can be used, as long as the amount of antibody, antigen, or antigen-antibody complex corresponding to the amount of antigen in the solution to be tested (particularly the amount of Aβ3pE or a fragment thereof) is detected by chemical or physical means and calculated from a standard curve obtained by using a standard solution containing a known amount of antigen. For example, turbidimetry, competitive methods, immunoassays, and sandwich methods are suitable. In terms of sensitivity and specificity, the sandwich method is particularly preferred.

[0193] In the sandwich method, a test solution is reacted with an insoluble antibody (such as an insoluble anti-Aβ3pE antibody) (first reaction), and a labeled second antibody is reacted (second reaction). The activity of the labeling agent on the insoluble carrier is then measured, thereby determining the amount of Aβ3pE or its fragment in the test solution. The first reaction and the second reaction can be performed simultaneously or sequentially.

[0194] In the measurement method, a labeling substance, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used as a labeling agent. Examples of radioisotopes include 125 1. 131 I. 3 H and 14 C. Enzymes are typically made detectable by conjugation to an appropriate substrate that then catalyzes a detectable reaction. Examples include, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase, preferably horseradish peroxidase. The luminescent material includes, for example, luminol, luminol derivatives, luciferin, aequorin, and luciferase. In addition, the avidin-biotin system can also be used to label the antibodies and immunogens of the present invention. When the immunogen or antibody is insoluble, physical adsorption or chemical binding, which is commonly used for insolubilization or immobilization of proteins or enzymes, can be used. Examples of carriers include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicone polymers, and glass.

[0195] In another embodiment for detecting or diagnosing β-amyloid related diseases, a biological sample including tissues, body fluids such as cerebrospinal fluid (CSF), blood, plasma, serum, urine, etc. is contained and contacted with an appropriate amount of a first antibody to produce an immune complex. This contact generally involves adding the sample to a solid matrix coated with the first antibody. The fluoride produced by contacting the sample with the second antibody is separated from the sample by elution. However, other recovery methods can be used. The recovered complex is contacted with at least one second antibody, which is directed against an antigenic determinant on the antigen and is capable of binding to the antigen on the complex. The antigenic determinant targeted by the second antibody can be the same antigenic determinant targeted by the first antibody, due to the multi-epitope characteristics of the antigenic entity. Any of the above-mentioned labels can be used to make the first antibody or the second antibody detectable. In a preferred embodiment, the second antibody is made detectable. The presence of a detectable antibody bound to the complex can be easily detected using techniques known in the art, and the complex consists of an antigen bound to the first antibody and the second antibody. By comparing the results obtained in the biological sample with those obtained on the control sample, the presence or level of the altered Aβ3pE or its fragment can be determined.

[0196] In vivo methods

[0197] Aspects of the present invention relate to methods for preventing, ameliorating, treating and / or reducing amyloid-β deposition in amyloid-β-related conditions, comprising administering an antibody or antigen-binding fragment thereof as disclosed herein to a subject of the present invention in a therapeutically effective amount. Additional aspects of the present invention include pharmaceutical compositions for preventing, ameliorating, treating and / or reducing amyloid deposition in amyloid-β-related conditions, comprising an antibody or antigen-binding fragment thereof as disclosed herein. The methods of the present invention comprise administering an effective amount of one or more antibodies or antigen-binding fragments thereof as described herein to a subject in need thereof.

[0198] In one aspect, the present invention relates to a method for preventing, ameliorating, treating and / or reducing amyloid-β deposition in a disorder characterized by the formation of plaques containing β-amyloid in a human, the method comprising administering to a human in need of such treatment, preferably peripherally, a therapeutically effective amount or a prophylactically effective amount of an antibody according to the present invention or an immunologically reactive fragment thereof, the antibody specifically binding to human Aβ3pE. In another aspect, the present invention relates to a method for inhibiting the formation of amyloid plaques and / or a method for clearing amyloid plaques in a human, the method comprising administering to a human subject in need of such inhibition or clearance an effective amount of an antibody according to the present invention, wherein the antibody sequesters Aβ3pE peptides in the brain and induces clearance of altered Aβ3pE in the brain. In additional aspects, the present invention relates to such humanized antibodies, including immunologically effective portions thereof, and methods for their preparation.

[0199] A subject in need thereof is a human suffering from or susceptible to a disorder characterized by the formation of plaques comprising beta-amyloid. In one embodiment, the disorder is Alzheimer's disease. In other embodiments, the disorder is dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, or hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D).

[0200] Humanized antibody is the antibody from non-human species, and its protein sequence has been modified to increase the similarity of the naturally occurring antibody variants in them and people. Generally speaking, the protein sequence of humanized antibody is substantially the same as the protein sequence of human variant, except that its complementary determining region (CDR) fragment is responsible for antibody binding to some or all of the non-human sources of the ability of its target antigen. The framework region of variable region is replaced by corresponding people's framework region, retaining non-human CDR is substantially intact. In some cases, humanized antibody has a small amount of replacement in one or more in non-human CDR region, to retain binding affinity and / or the dissociation constant of non-human antibody.

[0201] Humanized antibodies also refer to antibodies comprising a human framework, at least one CDR from a non-human antibody, and any constant regions present therein that are substantially identical to human immunoglobulin constant regions, i.e., at least about 85%, 90%, preferably at least 95% identical or 98% identical. Thus, except for one or more of the CDRs, all parts of a humanized antibody are substantially identical to corresponding parts of a human immunoglobulin sequence. For example, a humanized immunoglobulin will generally not encompass chimeric mouse variable region / human constant region antibodies.

[0202] Humanized antibodies have at least three potential advantages over nonhuman and chimeric antibodies for human therapy: 1) because the effector portion is human, it can better interact with other parts of the human immune system (e.g., activating microglia to clear plaques); 2) the human immune system should not recognize the framework or C regions of the humanized antibody as foreign, so the antibody response to such administered antibodies should be less than that to completely foreign nonhuman antibodies or partially foreign chimeric antibodies; and 3) the half-life of administered nonhuman antibodies in human circulation has been reported to be shorter than that of human antibodies.

[0203] In the method for treating and preventing plaques characterized by the formation of beta-amyloid, the antibody of the present invention or its antigen-binding fragment (including immunologically active fragment) is administered to a subject at risk of or exhibiting amyloid beta-related symptoms or pathology, such as clinical or preclinical Alzheimer's disease, dementia associated with Down syndrome, or clinical or preclinical amyloid angiopathy using standard administration techniques. Preferably, peripheral administration (i.e., not by administration to the central nervous system) is performed by intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, oral, sublingual or suppository administration. Although the antibody or its binding fragment can be directly administered into the ventricular system, cerebrospinal fluid or brain parenchyma, and the technology for accessing these locations is well known in the art, it is not necessary to use these more difficult procedures. When administered by simpler techniques that rely on the peripheral circulatory system, the antibody of the present invention or its binding fragment is effective. Advantages of the present invention include the ability of the antibody or its antigen-binding fragment to exert its beneficial effects even if it is not directly provided to the central nervous system itself.

[0204] Pharmaceutical compositions for administration are designed to suit the selected mode of administration and appropriately use pharmaceutically acceptable excipients such as dispersants, buffers, surfactants, preservatives, solubilizers, isotonic agents, stabilizers, etc. Remington's Pharmaceutical Science, Mack Publishing Co., Easton Pa., latest edition, incorporated herein by reference, provides an overview of formulation techniques generally known to practitioners.

[0205] It may be particularly useful to alter the solubility properties of an antibody of the invention to render it more lipophilic, for example by encapsulating it in liposomes or by blocking polar groups.

[0206] Preferably, the drug is delivered systemically via a peripheral route of intravenous, intraperitoneal or subcutaneous injection. Suitable carriers for such injections are very simple. However, in addition, administration can also be performed via mucosal nasal aerosols or suppositories. Formulations suitable for such modes of administration are well known and typically include surfactants that facilitate transmembrane transfer. Such surfactants are typically derived from steroids or cationic lipids, such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA) or various compounds, such as cholesterol hemisuccinate, phosphatidylglycerol, etc.

[0207] In one embodiment, the humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 100% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 100% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more. The humanized antibody of the present invention is administered in an amount of 0.1% by weight or more.

[0208] For antibody administration, dosage range is about 0.0001mg / kg to 100mg / kg of host body weight, preferably 0.01mg / kg to 75mg / kg. For example, dosage can be 0.02mg / kg, 0.25mg / kg, 0.5mg / kg, 0.75mg / 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, 60mg / kg, 65mg / kg, 70mg / kg, or 75mg / kg of host body weight. In embodiments, the dosage is in the range of 0.01 mg / kg to 10 mg / kg, or in the range of 0.1 mg / kg to 15 mg / kg, or in the range of 0.1 mg / kg to 20 mg / kg, or in the range of 0.1 mg / kg to 30 mg / kg, or in the range of 0.1 mg / kg to 40 mg / kg, or in the range of 0.1 mg / kg to 50 mg / kg, or in the range of 0.1 mg / kg to 60 mg / kg, preferably at least 1 mg / kg, at least 5 mg / kg, at least 10 mg / kg, at least 20 mg / kg, at least 30 mg / kg, at least 40 mg / kg, at least 50 mg / kg, or at least 60 mg / kg. In a preferred example, the dosage can be about 10 kg / mg, about 20 kg / mg, about 30 kg / mg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, or about 70 mg / kg. In a particularly preferred example, the antibody is administered intraperitoneally in a dose range of about 0.3 mg / kg to about 60 mg / kg. In an exemplary treatment regimen, the antibody is administered intraperitoneally in a dose of about 10 kg / mg, about 20 kg / mg, about 30 kg / mg, about 40 mg / kg, about 50 mg / kg, or about 60 mg / kg.

[0209] As used herein, the term "about" when referring to a measurable value such as an amount is meant to encompass variations from the specified value of ±20% and ±0.1%, preferably ±15% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.5%, ±0.1%, 0.05% or 0.01%, as such variations are appropriate.

[0210] Exemplary treatment regimens require administration once every two weeks or once per month or once every 3 to 6 months. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the indicated range. Antibodies are typically administered on multiple occasions. The time interval between single doses can be weekly, monthly, or annually. Intervals can also be irregular, as indicated by measuring the blood levels of antibodies to A β in the subject. Alternatively, the antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the antibody in the patient. Generally speaking, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies.

[0211] The dosage and frequency of administration may vary depending on whether the treatment is preventive or therapeutic. In preventive applications, a relatively low dose is administered at relatively infrequent intervals over a longer period of time. Some subjects continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dose at relatively short intervals may be required until the progression of the disease slows or terminates, and preferably until the subject shows partial or complete improvement in the symptoms of the disease. A preventive mechanism may then be employed.

[0212] In some methods, the dose is administered to achieve a plasma antibody concentration of 1 μg / ml to 1000 μg / ml, and in some methods 25 μg / ml to 300 μg / ml. Alternatively, the antibody may be administered as a sustained-release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the antibody in the subject.

[0213] Treatment with an antibody of the invention may be a stand-alone therapy. Alternatively, treatment with an antibody of the invention may be a component or phase of a combination therapy regimen in which one or more additional therapeutic agents are also used to treat the individual.

[0214] When used for in vivo therapy, the antibodies of the present invention or their antigen-binding fragments are administered to an individual in a therapeutically effective amount, e.g., to reduce, remove or prevent β-amyloid plaques or to improve the cognitive function of a subject suffering from AD or other β-amyloid-related diseases. The antibodies or their antigen-binding fragments are administered to an individual by known methods, such as intravenous administration, e.g., bolus injection or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes. The medicament of the present invention may optionally be administered in combination with other medicaments that are at least partially effective for treating amyloidosis. In the case of Alzheimer's disease and related conditions in which amyloid deposition occurs in the brain, the antibodies of the present invention or their antigen-binding fragments may be administered in combination with other medicaments that enhance the medicament of the present invention to cross the blood-brain barrier.

[0215] In one embodiment of the present invention, the antibodies or antigen-binding fragments thereof of the present invention bind to 3pE Aβ in plaque deposits. By binding to 3pE Aβ in dental plaque deposits, the antibodies or antigen-binding fragments thereof can induce plaque removal. Plaque removal can be induced by activating microglia surrounding the plaque and destabilizing the plaque by removing stable Aβ forms. In addition, the antibodies or antigen-binding fragments thereof of the present invention can inhibit the plaque-seeding activity of 3pE Aβ. Compared to vascular amyloid, the potential enrichment of 3pE Aβ in plaques may increase the therapeutic safety window of immunotherapy.

[0216] Kits and devices

[0217] The present invention provides kits and devices that can be used in the above-described methods. Preferably, the kits and devices comprise an antibody or antigen-binding fragment thereof that binds to 3pE Aβ. In addition, the kits may comprise reagents and instructional materials. The instructions may be, for example, printed on paper and / or provided on an electronically readable medium. Alternatively, the instructions may be provided by directing the user to an internet website, such as one designated by the manufacturer or distributor of the kit.

[0218] The reagents contained in the kits of the present invention may be provided in various container forms such that the activities of the various components are substantially maintained and the components themselves are not substantially adsorbed or altered by the material of the container.

[0219] In one embodiment, the kit or device comprises an antibody or antigen-binding fragment thereof of the invention, preferably a purified antibody, more preferably a monoclonal antibody, even more preferably an isolated monoclonal antibody that binds to 3pE Aβ peptide. In an embodiment, the antibody is expressed by a hybridoma cell.

[0220] Implementation Plan

[0221] Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the following polypeptide sequence:

[0222] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0223] b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively;

[0224] c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively;

[0225] d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively;

[0226] e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively;

[0227] f. SEQ ID NOs: 56, 57, 3, 4, 5 and 6, respectively;

[0228] g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively;

[0229] h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively;

[0230] i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively;

[0231] j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively;

[0232] k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively;

[0233] l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or

[0234] m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively;

[0235] The antibody or antigen-binding fragment thereof specifically binds to 3pE Aβ, preferably human 3pE Aβ.

[0236] Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 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 NO: 10, 12, 14, 18, 22, 53 or 55.

[0237] Embodiment 3 is the isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, comprising:

[0238] a. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0239] a light chain variable region of the polypeptide sequence of 22;

[0240] b. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 9 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0241] 10;

[0242] c. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 11 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0243] A light chain variable region of the polypeptide sequence of 12;

[0244] d. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 13 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0245] A light chain variable region of the polypeptide sequence of 14;

[0246] e. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 15 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0247] A light chain variable region of the polypeptide sequence of 14;

[0248] f. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 16 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0249] A light chain variable region of the polypeptide sequence of 14;

[0250] g. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 20 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0251] A light chain variable region of the polypeptide sequence of 14;

[0252] h. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 17 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0253] A light chain variable region of the polypeptide sequence of 18;

[0254] i. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 19 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0255] A light chain variable region of the polypeptide sequence of 18;

[0256] j. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0257] 53; or

[0258] k. A heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a heavy chain variable region having a polypeptide sequence of SEQ ID NO:

[0259] The light chain variable region of the polypeptide sequence of 55.

[0260] Embodiment 4 is the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein the antibody or antigen-binding fragment thereof is chimeric.

[0261] Embodiment 5 is the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment thereof is human or humanized.

[0262] Embodiment 6 is an isolated monoclonal antibody comprising:

[0263] a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0264] b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0265] c. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 52; or

[0266] d. A heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 54.

[0267] Embodiment 7 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6.

[0268] Embodiment 8 is a vector comprising the isolated nucleic acid according to embodiment 7.

[0269] Embodiment 9 is a host cell comprising the vector according to embodiment 8.

[0270] Embodiment 10 is a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 6 and a pharmaceutically acceptable carrier.

[0271] Embodiment 11 is a method of treating a condition associated with the formation of plaques containing β-amyloid in a subject in need thereof, the method comprising administering to the subject in need thereof the monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6 or the pharmaceutical composition according to embodiment 10.

[0272] Embodiment 12 is a method according to embodiment 11, wherein the condition is Alzheimer's disease.

[0273] Embodiment 13 is a method according to embodiment 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 amyloidosis (HCHWA-D).

[0274] Embodiment 14 is a method of reducing plaques associated with Alzheimer's disease in a subject in need thereof, the method comprising administering to the subject in need thereof the monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6 or the pharmaceutical composition according to embodiment 10.

[0275] Embodiment 15 is a method of preventing the vaccination activity of 3pE Aβ in a subject in need thereof, the method comprising administering to the subject in need thereof the monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6 or the pharmaceutical composition according to embodiment 10.

[0276] Embodiment 16 is a method of producing the monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions in which the monoclonal antibody or antigen-binding fragment thereof is produced, and recovering the antibody or antigen-binding fragment thereof.

[0277] Embodiment 17 is a method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, the method comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0278] Example

[0279] The invention can be further understood in light of the following non-limiting examples.

[0280] Example 1: Monoclonal Antibody Generation and Humanization Process

[0281] Three Balb / c mice (Janvier Labs) were vaccinated with H2N-pEFRHDSGC-COOH (Eurogentec) (SEQ ID NO: 47) in complete Freund's adjuvant (Sigma; St. Louis, MO). Peptides were prepared by coupling the peptides via the COOH-terminal cysteine residue to bovine serum albumin activated with maleimide (Life Technologies; Carlsbad, CA) using commercially available kits, such as the Imject Maleimide Activated BSA kit (Pierce; Rockford, IL), according to the manufacturer's instructions. Mice were boosted every two weeks with 100 μg or 200 μg of BSA-coupled peptide, first in complete Freund's adjuvant and then in incomplete Freund's adjuvant.

[0282] Hybridoma and Antibody Production: Mice displaying the highest serum titers were selected for fusion, while spleens from other mice were isolated and frozen in liquid nitrogen. On day 4, prior to fusion or spleen extraction, all mice were boosted intraperitoneally with 100 μg of H2N-pEFRHDSGC-COOH (SEQ ID NO: 47) conjugated to BSA (Merck; Kenilworth, NJ) in saline. Mouse spleen cells were fused with SP2 / 0 cells (ATCC; Manassas, VA) using a modified procedure of Kohler and Milstein (Euro. J. Immunol., 1976; 292-295). Hybridomas were plated in 30×96-well plates and screened 10 days later in a direct ELISA against 0.5 μg / well of unconjugated Aβ3pE-40 peptide (AnaSpec; Fremont, CA). Positive cells were tested for (lack of) cross-reactivity to 0.5 μg / ml coated Aβ 1-40 peptide (AnaSpec) and immediately subcloned.

[0283] After fusion, 17 clones reacted positive with human Aβ3pE-40 synthetic peptide (SEQ ID NO: 40) in direct-plate ELISA screening and were frozen in liquid nitrogen.

[0284] All hybridomas were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Europe), Hybridoma Fusion Cloning Supplement (2%) (Roche; Brussels, Belgium), 2% HT (Sigma), 1 mM sodium pyruvate, 2 mM L-glutamine, and penicillin (100 U / ml) and streptomycin (50 mg / ml). All products are commercially available and purchased from Life Technologies. Cells were incubated in a humidified 8% CO2 air incubator.

[0285] Direct ELISA for antibody selection: The screening ELISA for detecting the above-mentioned Aβ3pE-40 antibodies was a direct ELISA in which 0.5 μg / ml free human Aβ3pE-40 peptide (SEQ ID NO: 40) was coated in 50 μl / well coating buffer (10 mM Tris, 10 mM NaCl and 10 mM NaN3, pH 8.5) in NUNCMaxisorp (Life Technologies) flat-bottom high-binding 96-well microtiter plates at 4°C overnight.

[0286] The next day, the plates were blocked with 75 μl / well of 0.1% casein (Merck) in PBS for 60 minutes at room temperature to reduce nonspecific binding. Next, 50 μl of hybridoma supernatant was added and incubated at 37°C for 1 hour. After washing, bound monoclonal antibodies were detected at 37°C for 1 hour using 50 μl / well of sheep anti-mouse IgG conjugated to horseradish peroxidase (Amersham-Pharmacia Biotech; Little Chalfont, United Kingdom). Both reagents were diluted in 0.1% casein / PBS. The plates were washed and 50 μl of 0.42 mM 3,5,3',5'-tetramethylbenzidine (Biorad), 0.003% (volume / volume) H2O2 (Biorad) in 100 mM citric acid (Biorad; Hercules, CA), 100 mM sodium dihydrogen phosphate (pH 4.3) (Biorad) were added as substrates. The reaction was allowed to proceed for a maximum of 15 minutes at room temperature on a plate shaker, after which color development was stopped with 50 μl / well of 2N H2SO4 and the plate was read at 450 nm on a microtiter plate reader (Thermomax, Molecular Devices). The cross-reactivity of the selected monoclonal antibodies to full-size free human Aβ1-40 was tested using the same direct ELISA as the screening assay.

[0287] From 17 Aβ3pE-40 reactive clones, BAMB31_1 was selected based on affinity and selectivity for further characterization (see Examples 2 and 3). This antibody was determined to have a mouse IgG1 isotype heavy chain and a mouse kappa light chain. Although mouse IgG1 Fc has only 70% sequence identity and 76% sequence similarity to mouse IgG2a Fc, these isotypes have different activities and protein profiles. Compared to mouse IgG2a, mouse IgG1 has less mouse Fc effect and complementary function due to weaker binding to mouse FcγRI, FcγRIII and FcγRIV receptors and mouse C1q. Mouse IgG2a is considered to be the isotype closest to human IgG1 activity, binding to mouse FcγRI, FcγRIII and FcγRIV receptors and mouse C1q, thereby having complement, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) activities that can contribute to the clearance of Aβ plaques.

[0288] The sequence of the BAMB31 heavy chain was altered from mouse IgG1 to mouse IgG2a to form BAMB31_2a. The retention of reactivity after V region cloning was confirmed using SPR as described below.

[0289] Humanization process: The parent antibody BAMB31-2a (mIgG2a) was humanized using a procedure similar to that of Singh et al. (MAbs 2015; 7(4): 778-91), except that CDR-H2 was described according to the AbM definition used for this work (Martin, AC, PNAS 86: 9268-9277, 1989). Briefly, complementary determining regions (CDRs) were identified in the mouse parent sequence. These sequences were compared with human germlines, and four human germline heavy chains and two human germline light chain frameworks in which the mouse CDRs were transplanted were selected. The human J segments of the VL and VH of each parent antibody were selected by comparing the mouse and human J segment sequences to maximize sequence identity. Molecular models of the Fv regions of the parent mAbs were generated in MOE (CCG; Montreal, Canada) using default parameters. The resulting models were graphically inspected to identify framework positions that may be important for binding and / or antibody stability. Obtain antibody library, wherein these positions also have people / mouse binary combination except the CDR of transplantation.In the library, each chain with the mouse CDR of transplantation is paired with relative mouse parent chain.Like this, only have one chain to be suitable for people's framework, and based on antigen binding, determine back mutation.Then humanized VH and VL are combined to obtain final candidate antibody.

[0290] Library clones were expressed as Fabs in E. coli and tested for peptide binding by ELISA, with signals compared to the fully murine parental molecule. Molecules showing binding greater than 80% of the murine parental signal were selected for sequencing. The sequences were analyzed, and a human-adapted heavy chain and a human-adapted light chain were selected for combination and expression as a monoclonal human IgG1 antibody. For each antibody, all VH / VL humanized pairs were expressed, purified, and evaluated for antigen binding, Epivax computer simulations for immunogenicity risk, the number of residues restored to the mouse sequence, and biophysical properties.

[0291] Results: Some residues needed to be reverted to the mouse sequence to maintain parental binding. Retention of binding, Epivax in silico immunogenicity risk, and biophysical properties were not dependent on the number of reverted mouse sequences, but rather on the position of the reverted mouse sequence. Representative HFA mAb characterization results from this analysis are shown in Tables 2 and 3.

[0292] Table 2: Example of BAMB31HFA characterization results

[0293]

[0294] BAMB246 parental human IgG1 chimera

[0295] Values outside the expected range are in bold: Total number of residues recovered to the mouse sequence > 5; Hc and Lc Epivax risk score > -10; V region combination Epivax risk score > -20; SEC% monomer value < 95% kd (1 / s) value > 1.00E-04; KD (M) value > 2.50E-11; T start (℃) < 60; Tm1 (℃) < 65; Tagg (℃) < 65.

[0296] Reduction of post-translational modification risk: The parental antibody and the human framework-adapted variants contain an N post-translational deamidation modification motif in HCDR2. To address this potential issue, separate libraries were created for N and G residues using degenerate oligonucleotides. These generated new sequences randomly introduced all 20 amino acids at each position. Each library was screened for retained binding. Variant antibodies that exhibited similar binding to the murine parent were selected for sequencing.

[0297] Results: After sequencing, both N to S and N to G mutants had comparable binding to the parent. The lead HFA variant was cloned and expressed as wild-type IgG1 (BAMAB674), and an IgG1 with M37Y, S39T, and T41E point mutations in the Fc region (based on the Hc constant region Fc IgG1 heavy chain constant region sequence gene bank accession number AEV43323 numbering) was named +YTE IgG1 (BAMB675). These mutations are known to increase affinity for FcRn and extend circulation half-life (Properties of Human IgG1s Engineered for Enhanced Binding to the Neonatal Fc Receptor (FcRn), Dall'Acqua WF., JBC, 2006). The characterization of BAMB674 and BAMB675 is shown in Table 3.

[0298] Table 3: Characterization of BAMB674 and BAMB675

[0299] Properties BAMB674 BAMB675 Isotype wt IgG1 +YTE IgG1 HkDJ IGHV1-46*03 IGHV1-46*03 Hc FW positions requiring mouse residues M48I, M70L M48I, M70L Hc CDR2 NG motif reduction N55S N55S LqCy IGKV2-30*01 IGKV2-30*01 Lc FW positions requiring mouse residues V109L V109L Lc CDR1 NG motif reduction NG→RA NG→RA KD (pM) 25.4 25.7 Tm start 62.9℃ 57.7℃ Tm1 67.3℃ 64.1℃ Tagg 68.4℃ 67.7℃ SEC% monomer 97 98 High concentration stable for >2 weeks >100mg / ml >100mg / ml

[0300] Table 3a: Thermal stability

[0301]

[0302] *N=1

[0303] Example 2: Thermal stability test

[0304] For the variants adapted to the BAMB31 human framework, their thermostability was assessed using nanometer differential scanning fluorimetry (NanoDSF) to measure melting onset temperature (T), first melting transition temperature (Tm1), and initial aggregation detection temperature (T). The data for some variants are shown in Table 2 (columns 6 to 8) and Table 3 (rows 10 to 12) and Table 3a.

[0305] Materials and methods: The thermal stability of the samples was determined using an automated Prometheus instrument. Measurements were performed by loading samples from a 384-well sample plate into a 24-well capillary tube. Replicate runs were performed for each sample. The Prometheus NanoDSF user interface (Melting Scan tab) was used to set the experimental parameters for the run. The thermal scan range for a typical IgG sample was 20°C to 95°C at a rate of 1.0°C / min. The typical concentration of the sample was 0.3 mg / mL to 1 mg / mL. The intrinsic fluorescence of the molecule at 330 nm and 350 nm was used to monitor the unfolding during the temperature ramp and was recorded as the change in fluorescence intensity over time. This is called the thermal scan result (Tm). Using backreflection technology in parallel, the instrument calculates the onset aggregation temperature (Tagg) during the thermal ramp. Therefore, the NanoDSF method is able to simultaneously measure the conformational and colloidal stability of lead candidates, which are often monitored as indicators of long-term sample stability under varying conditions.

[0306] Example 3: Epivax In silico Immunogenicity Risk Assessment

[0307] EpiMatrix software (EpiVax Inc.) for predicting MHC class II binding was used to perform computer simulation analysis of the anti-Aβ3pE mab V region. The software examines consecutive amino acid 9-mers to identify potential HLA class II binding sequences. The database includes the most common HLA types covering approximately 95% of the population. If the HLA receptor of an antigen-presenting cell binds to a peptide antigen restriction site, the other side of the peptide (epitope) can bind to T effectors or T regulatory cells, which in turn can stimulate or inhibit an immune response to a protein with the epitope. The software generates an antigen restriction site binding score that can be adjusted for predicted T regulatory cell binding. The score is normalized relative to the size of the protein and the number of binding events that result in an output indicating the predicted immunogenicity of the protein.

[0308] Example 4: Analytical characterization of purified mAb

[0309] The protein concentration of each purified mAb was determined by measuring the absorbance at 280 nm on a NanoDrop 1000 spectrophotometer or a Trinean Drop Sense 96 multichannel spectrophotometer and calculated using the extinction coefficient based on the amino acid sequence.

[0310] SE HPLC of the purified antibody was performed in the following manner: on a TOSOH TSKgel BioAssist G3SWxl column, the sample was run on a Waters Alliance HPLC at a rate of 1 mL / min in 0.2 M sodium phosphate at pH 6.8 for 20 minutes. The column effluent was monitored by absorbance at 280 nm. The results are shown in Tables 2 and 3.

[0311] Example 5: Binding kinetics and affinity measurements

[0312] Surface plasmon resonance (SPR) is a label-free detection method for studying biomolecular interactions. By monitoring small changes in mass on the sensor surface, this direct real-time binding assay provides qualitative and quantitative data on the interactions between biomolecules; i.e., determining the equilibrium binding constant (affinity, K D ) and the kinetic rate constant (k a / k d ; Complex association rate k a , and the complex dissociation rate k d This method can be used to study protein-protein and protein-nucleic acid interactions, as well as interactions between proteins and small molecules. Here, the interactions between 3pE-specific antibodies and human Aβ3pE-40 (SEQ ID NO: 40) or Aβ3pE-28 (SEQ ID NO: 42), human Aβ1-40 (SEQ ID NO: 41) or Aβ1-28 (SEQ ID NO: 43), and rodent Aβ3pE-28 peptides (SEQ ID Nos: 45 and 46) were studied.

[0313] Materials and methods

[0314] SPR: Affinity studies of BAMB31-2a (mIgG2a) for Aβ-3pE-40 peptide (SEQ ID NO: 40) were performed using a mouse antibody capture kit from GE Healthcare. J&JPRD / Aβ / pE3 / 1mIgG2a (described in U.S. Patent Publication No. 2018 / 0142011) and mE8c mIgG2a (described in US9944696 and US8679498) were included as control antibodies. Immobilization of anti-mouse antibodies was performed on a CM5 sensor chip via amine coupling according to the manufacturer's protocol. Subsequently, the antibody of interest (1 μg / ml) was captured by anti-mouse antibody to a level of 300 RU, after which various concentrations (3.125 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM) of diluted human Aβ3pE-40 peptide (SEQ ID NO: 40) were injected into the running buffer (containing 2.7 mM KCl, 137 mM NaCl, and 0.05% surfactant P20 (Tween TM The surface was regenerated with 10 mM glycine HCl at pH 1.7 for at least 180 seconds and an additional 60 seconds. Human Aβ(1-40) peptide (SEQ ID NO: 41) was used as a negative control.

[0315] Using the Optical Biosensor T200 Affinity measurements were performed. Kinetic analysis was performed based on a 1:1 binding fitting model using Biacore T200 evaluation software (version 2.0).

[0316] In some cases, the binding affinity and specificity of anti-Aβ3pE mAbs were measured by SPR using different instruments (Biacore T200, Biacore 8K or MASS-2 (Biacore, Inc.) and anti-human or anti-mouse immunoglobulin biosensor surfaces. Anti-human or anti-mouse immunoglobulin antibodies were covalently coupled to the surface of CM4 or CM5 sensor chips (GE Healthcare) using the manufacturer's instructions for amine coupling chemistry. The antibody of interest was captured on an anti-human or mouse immunoglobulin sensor chip to a level of 300RU to 400RU, and then various concentrations of Aβ peptide or protein (examples: human Aβ3pE-40 (SEQ ID NO: 40), Aβ3pE-28 (SEQ ID NO: 42), scrambled Aβ3pE-28 (SEQ ID NO: 44), Aβ1-28 (SEQ ID NO: 43), mouse Aβ3pE-28 (SEQ ID NO: 46), or fibronectin) were injected into a 4% PBS containing 0.005% surfactant P20 (Tween 100). TM 20) in HEPES buffered saline. 30 μL of 10mM Gly pH 1.5 were pulse-injected twice at a rate of 100 μL / min to regenerate the surface. The reported data are the differences in SPR signals between the flow cell containing the captured antibody and the reference cell without the captured antibody. The additional instrument contribution to the signal was removed by subtracting the data of the blank injection from the signal deducted from the reference. When applicable, the data were analyzed by using Biaevaluation software (Biacore, Inc.) with a 1:1 binding model fitting the association phase and dissociation phase (global fitting) at all concentrations. Otherwise, the data were qualitatively assessed as yes / no binding.

[0317] Immunohistochemistry of formalin-fixed paraffin-embedded brains: For immunohistochemical analysis, after dewaxing and rehydration of the sections, antigen retrieval was performed by incubating transgenic mouse brain slides in formic acid (70% distilled water) for 10 minutes, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (DAKO; Glostrup, Denmark, S2023). Sections were incubated with different concentrations (working concentrations: 2 μg / ml, 0.1 μg / ml to 0.05 μg / ml to 0.025 μg / ml in antibody diluent containing background reduction components (DAKO, S3022)) of BAMB674, BAMB675, hE8L, R17L, R17, CI-C7, B12L, Antibody I, or Antibody II (the latter seven antibodies were previously described in US9944696B2 and US8679498B2) for 1 hour. After extensive washing, HRP-conjugated anti-human secondary antibody (PI-3000, Vector labs; Burlingame, CA, 1 / 500 in antibody diluent (DAKO, S0809)) was applied to the slides for 1 hour, followed by development with 3,3-diaminobenzidine (DAB) (DAKO, K3468). The slides were counterstained with hematoxylin, dehydrated, and permanently mounted with Vectamount (H-5000, Vector Labs).

[0318] Results: Kinetic analysis of the monoclonal antibody BAMB31_2a (mIgG2a) confirmed affinity binding to the Aβ3pE-40 peptide (SEQ ID NO: 40). No binding was detected when the human Aβ1-40 peptide (SEQ ID NO: 41) was applied at concentrations up to 50 nM. The sensorgram (single cycle kinetics) demonstrating the binding interaction of BAMB31_2a (mIgG2a) with the human Aβ3pE-40 peptide (SEQ ID NO: 40) is shown in Figure 1 As comparator molecules, J&JPRD / Aβ / pE3 / 1mIgG2a and mE8cmIgG2a ( Figure 2 ) and confirmed that BAMB31 has a higher affinity than mE8c and J&JPRD / Aβ / pE3 / 1. D ) and the kinetic rate constant (k a / k d ) are shown in Table 4.

[0319] Table 4: Kinetics of J&JPRD / Aβ / pE3 / 1, BAMB31, and mE8cmIgGa

[0320]

[0321] Kinetic analysis of BAMB31 HFA mAbs (BAMB674 and BAMB675) with reduced risk of Hc CDR2 NG deamidation showed that they retained binding affinity for the Aβ3pE-28 peptide (SEQ ID NO: 42) relative to the BAMB31_2a (mIgG2a) parent and the BAMB246 (human IgG1 chimera) parent. D ) and the kinetic rate constant (k a / k d ) are shown in Table 5.

[0322] Compared to the previously described humanized 3pE-specific antibodies hE8L, R17L, R17, CI-C7, B12L, Antibody I and Antibody II (previously described in US9944696B2 and US8679498B2), the current BAMB31 HFA molecules have higher affinity as shown in Table 5.

[0323] Table 5: 3pEAβ binding kinetics of BAMB31HFA and mE8cHFA molecules

[0324]

[0325] Human chimeric antibodies have mouse variable regions within human IgG1 constant regions.

[0326] Table 5a: Binding affinity comparison

[0327] sample ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> BAMB700 2.88E+06 9.86E-06 3.42E-11 BAMB701 3.01E+06 9.47E-05 3.14E-11 BAMB674 3.39E+06 1.36E-04 4.01E-11 BAMB675 3.74E+06 1.66E-04 4.43E-11 Control mAb 4.11E+06 1.56E-04 3.79E-11

[0328] HU-3pE-β-amyloid concentration range 0.6nM to 4.5nM

[0329] The higher affinity measured by SPR also translated into improved plaque binding. A dilution series of the primary antibody was performed on brain sections of transgenic mouse brains by immunohistochemistry. At a concentration of 2 μg / mL, all antibodies gave visible plaque labeling, but to varying degrees. At a concentration of 0.1 μg / mL, antibodies C1 to C7 and R17L did not produce visible plaque labeling, based on their low affinity as measured by SPR. At a concentration of 0.05 μg / mL, BAMB674 and BAMB675 showed plaque labeling, while for the comparative molecules, there was no visual plaque labeling at this concentration ( FIG. 3 ). At a concentration of 0.025 μg / mL, there was (almost completely) no visible plaque labeling for all tested molecules.

[0330] In summary, the immunohistochemistry data confirmed the SPR data demonstrating that BAMB674 and BAMB675 have higher affinities compared to the comparator molecules.

[0331] BAMB246 (human IgG1 chimera) and current BAMB31 HFA mAbs (BAMB674 and BAMB675) have >3 log selectivity for the highly homologous mouse Aβ3pE-28 peptide (SEQ ID NO:46) and >5 log selectivity for fibronectin, Aβ1-28 peptide (SEQ ID NO:43), and amino acid 3-9 scrambled Aβ3pE-28 peptide (scrambled 3pE-28) (SEQ ID NO:44). Selectivity results and equilibrium binding constants (affinity, K) for related peptides and proteins D ) and the kinetic rate constant (k a and k a ) are shown in Table 6.

[0332] Table 6: Selective binding kinetics of BAMB246 (human IgG1 chimera) and HFA mAbs to relevant targets

[0333]

[0334] Scrambled 3pE-28 is a human Aβ3pE-28 peptide in which amino acids 3-9 are scrambled.

[0335] Fold selectivity was determined by dividing the KD in Table 4 by the KD in Table 3.

[0336] > 1.20E-06K D (M) No antigen binding was detected at the highest concentration tested, 1.2 μM.

[0337] The FcRn binding affinity of the BAMB31 HFA antibody to wild-type IgG1 (BAMB674) and +YTE IgG1 isotype (BAMB675) is shown in Table 7. Compared to wt IgG1 (BAMB674), the +YTE mAb (BAMB675) has approximately 3-fold higher affinity for both human and cynomolgus monkey FcRn, which translated into a longer circulating half-life of the +YTE antibody in cynomolgus monkey pharmacokinetic studies ( Figure 11 ).

[0338] Table 7: Comparison of FcRn binding affinity of wild-type HFA mAbs and +YTE IgG1

[0339]

[0340] KD(M) values are the average of 5 to 6 independent replicates.

[0341] Example 6: Sandwich ELISA for cross-reactivity testing

[0342] For the selected Aβ3pE monoclonal antibody BAMB31, cross-reactivity with rodent Aβ3pE-40 (SEQ ID NO: 45) and human Aβ1-40 (SEQ ID NO: 41), Aβ1-42 (SEQ ID NO: 48), Aβ11pE-40 (SEQ ID NO: 49), and Aβ11pE-42 (SEQ ID NO: 50) was assessed using synthetic peptides. The combination BAMB31 + JRF / cAβ40 / 28-HRPO was used to study cross-reactivity with Aβ1-40 (SEQ ID NO: 41), Aβ11pE-40 (SEQ ID NO: 49), and rodent Aβ3pE-40 (SEQ ID NO: 45), and the combination BAMB31 + JRF / cAβ42 / 26-HRPO was used to study cross-reactivity with Aβ1-42 (SEQ ID NO: 48) and Aβ11pE-42 (SEQ ID NO: 50). Concentrations up to 10,000 pg / mL were tested.

[0343] Materials and methods: The standards were dissolved in dimethyl sulfoxide (DMSO) (Sigma) at 0.1 mg / mL and stored at -80°C. For use in ELISA, the peptides were further diluted to 1 pg / mL in 0.1% casein in PBS. Ninety-six well plates (Maxisorb ELISA plates, NUNC) were coated overnight at 4°C with a monoclonal antibody from BAMB31_1 at a concentration of 1.5 μg / mL in coating buffer. The next day, the plates were washed and blocked with 0.1% casein in PBS for 1-4 hours at room temperature. The standards were incubated overnight at 4°C with a secondary antibody labeled with HRPO (JRF / cAβ40 / 28-HRPO or JRF / cAβ42 / 26-HRPO). After overnight incubation, the plates were washed and developed using a TMB peroxide EIA substrate kit (Biorad) according to the manufacturer's recommendations.

[0344] Results: BAMB31 showed selective binding to Aβ3pE-40 (SEQ ID NO:40) and Aβ3pE-42 (SEQ ID NO:51), and no cross-reactivity was detected with human Aβ1-40 (SEQ ID NO:41), human Aβ1-42 (SEQ ID NO:48), and rodent Aβ3pE-40 (SEQ ID NO:45), as well as human AβpE11-40 (SEQ ID NO:49) and AβpE11-42 (SEQ ID NO:50) at concentrations up to 10 ng / mL (Figure 4).

[0345] Figure 7 : Immunohistochemistry for detecting antibody reactivity against plaques in transgenic mouse and human AD brain tissue study

[0346] Antibody reactivity to plaques was studied in formalin-fixed, paraffin-embedded (FFPE) and cryopreserved brain tissue.

[0347] Materials and methods

[0348] Formalin-fixed paraffin-embedded brain: For immunohistochemical analysis, after dewaxing and rehydrating the sections, antigen retrieval was performed by incubating transgenic mouse brain slides in formic acid (70% distilled water) for 10 minutes, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (DAKO; Glostrup, Denmark, S2023). Sections were incubated with BAMB246 (huIgG1 chimera), BAMB674, or BAMB675 (working concentration: 4 μg / ml in antibody diluent (DAKO, S3022) containing background reduction components) for 1 hour. After extensive washing, HRP-conjugated anti-human secondary antibody (PI-3000, Vector labs, 1 / 500 in antibody diluent (DAKO, S0809)) was applied to the slides for 1 hour, followed by color development with 3,3-diaminobenzidine (DAB) (DAKO, K3468). Slides were counterstained with hematoxylin, dehydrated, and permanently mounted with Vectamount (H-5000, Vector Labs).

[0349] Cryopreserved Brain: Human brain samples were snap-frozen, cryostat-sectioned (20 μm thickness), and stored at −80°C until use. Sections were dried at room temperature before formalin fixation, blocked with endogenous peroxidase containing 3% hydrogen peroxide (DAKO, Glostrup, Denmark, S2023), and blocked for 1 hour with 1x PBS + 0.3% Triton X-100 and 10% normal goat serum (DAKO, X0907). The primary antibody pE3 / 16 (2 μg / ml in antibody diluent containing background reduction components (DAKO, S3022)) was applied to the sections for 1 hour. After thorough washing, slides were incubated with an HRP-conjugated anti-mouse secondary antibody (Envision, DAKO, K4000) and then labeled with DAB (DAKO, K3468). Slides were counterstained with hematoxylin, dehydrated, and mounted with organic mounting medium (Vectamount, Vector labs). Imaging was performed with a Hamamatsu Nanozoomer (Hamamatsu Photonics; Shizuoka, Japan).

[0350] Results: The reactivity of BAMB264 (human IgG1 chimera) and BAMB674 and BAMB675 was confirmed on FFPE tissues of transgenic mice. Figures 5A to 5F ). In addition, BAMB31_2a (mIgG2a) demonstrated significant plaque labeling in cryopreserved AD brain tissue (Figure 6). In frozen sections of human brain, most of the plaques detected by antibody 4G8 were also labeled with BAMB31 ( Figures 5A to 5F ).++

[0351] Example 8: Serum antibody levels after administration to transgenic mice

[0352] Serum antibody levels after treatment with BAMB31 and the comparator molecule mE8c were investigated.

[0353] Materials and Methods: Old transgenic mice (22 to 23 months old) expressing elevated levels of human Aβ42 and Aβ40 peptides received a single intraperitoneal (ip) injection of 20 mg / kg of mE8c mIgG2a, BAMB31-2a (mIgG2a), or isotype control antibody mIgG2a (n=5 / treatment group). Following antibody administration, whole blood was collected via the great saphenous vein at intermediate time points (24 and 48 hours after injection) and at the time of sacrifice (day 4 after injection) via orbital puncture. Collection tubes (100Z and 300Z, respectively; Sarstedt; Numbrecht, Germany). The collected whole blood was incubated at room temperature for 1 to 2 hours and then centrifuged at 10,000 rpm for 10 minutes at 4°C to separate the serum from the blood clot. Serum antibody levels were determined using an allotype-specific enzyme-linked immunosorbent assay (ELISA). For this purpose, Nunc MaxiSorp was coated with 1.5 μg / ml mouse monoclonal anti-IgG2a(a) (BD Biosciences; San Jose, CA) at room temperature. TM Flat-bottom plates (ThermoScientific; Waltham, MA) were plated and incubated overnight. Antibody standards for mE8cmIgG2a, BAMB31-2a (mIgG2a), and isotype control mIgG2a were prepared separately at a concentration of 1 μg / ml in blocking buffer (1% BSA in PBS + 0.05% Tween-20) and further diluted to 0.1 ng / ml in blocking buffer. After washing (PBS + 0.05% Tween-20), the samples were blocked with blocking buffer for 1 hour at room temperature. Next, the standards and pre-diluted serum samples were plated on a MAXISORP coated plate. TMThe plates were incubated at room temperature for 1 hour. After the sample incubation, the plates were washed and incubated at room temperature with peroxidase-AffiniPure goat anti-mouse IgG (Fcγ subclass 2a specific) antibody for 1 hour. The plates were washed and developed by adding TMB peroxidase EIA substrate kit (1 step, Pierce) to the wells. Development was stopped after 2 minutes by adding 2N H2SO4 to the wells and the plates were read at 450 nm using an EnVision multi-mode plate reader (Perkin Elmer).

[0354] Results: Serum antibody levels were measured 24 hours, 48 hours, and 4 days after intraperitoneal injection of 20 mg / kg antibody. The mean antibody concentrations in serum after 24 hours were comparable for all antibodies studied. For BAMB31_2a and the isotype control antibody, the antibody concentrations gradually decreased over time (up to approximately 30% decrease on day 4), while for mE8c a significantly higher decrease over time was observed, with a decrease of approximately 97% on day 4 ( Figure 7 ).

[0355] In conclusion, differences in the pharmacokinetic profiles of BAMB31_2a and mE8c following intraperitoneal injection in a mouse model of plaque deposition were demonstrated, indicating that clearance after treatment with the BAMB31_2a antibody was slower than that of mE8c.

[0356] Example 9: Chronic efficacy study in a transgenic mouse model

[0357] The efficacy of reducing amyloid burden and the effect on microbleeds after long-term treatment with BAMB31-2a mIgG2a were investigated in a transgenic mouse model.

[0358] Materials and methods: PDAPP (V717F) transgenic mice (mean age was 18.3 months when the study began) were treated intraperitoneally weekly with BAMB31-2a antibodies (mIgG2a) at a dosage of 30 mg / kg for 12 weeks. A control group receiving mIgG2a isotype control antibody injection was included in the experiment. Animals were euthanized on the 7th day after the last intraperitoneal injection (mean age was 21.1 months at the end of the study). Before collecting tissue, mice were perfused with PBS. The left hemisphere (part 1: hippocampus, part 2: the remaining brain without hippocampus / cerebellum / brainstem) was cryopreserved for further biochemical analysis, while the right hemisphere was fixed overnight in a formalin-based fixative, then paraffin-embedded and sliced (5 μm) with a microtome.

[0359] To assess the effect of long-term treatment on amyloid burden, both biochemical and immunohistochemical analyses were performed. For biochemical analysis, brains were homogenized in ice-cold 5M guanidine hydrochloride and 50mM Tris / HCl extraction buffer (100mg tissue / ml extraction buffer) using Tallprep D lysis matrix tubes (MP Bio). After homogenization, samples were placed on an inverted rotating wheel at room temperature for 3 hours. The resulting homogenate was stored at -80°C prior to analysis by MSD sandwich immunoassay.

[0360] Synthetic Aβ peptide standards were dissolved in dimethyl sulfoxide (DMSO) (Sigma) at 0.1 mg / mL and stored at -80°C. For use in MSD immunoassays, the peptides were further diluted in 0.5 M GuHCl + 5 mM Tris-HCl - pH 8.0 (the extraction buffer was diluted 10-fold in 0.1% casein in PBS). The GuHCl extract was thawed and diluted 1:10 in an ice-cold 0.1% casein solution in PBS and centrifuged at 20,000 g for 20 minutes at 4°C. The supernatant was recovered for sandwich MSD assays, and the sample was further diluted in 0.5 M GuHCl + 5 mM Tris-HCl - pH 8.0 (the extraction buffer was diluted 10-fold in 0.1% casein in PBS).

[0361] 96-well sector plate standards (Meso Scale Discovery; Rockville, MD) were coated with monoclonal antibodies at a concentration of 1.5 μg / mL in PBS at 4°C overnight. The next day, the plates were washed and blocked with 0.1% casein in PBS for 2 hours at room temperature. The standards and samples were incubated with biotinylated secondary antibodies at 4°C overnight. After the overnight incubation, the plates were washed and incubated with secondary detection reagents (streptavidin-SULFO-TAG TM The plates were incubated with the markers at room temperature for 2 hours. The plates were washed and 2 times the reading buffer T was added, after which the plates were read according to the manufacturer's recommendations. 2 Aβ concentration was determined using a standard curve with a four-parameter logistic model with a weighting function.

[0362] The combination JRF / AβN / 25+4G8-biotin antibody was used to study the Aβ1-x concentration in brain homogenates.

[0363] For immunohistochemical analysis, after sections were deparaffinized and rehydrated, antigen retrieval was performed by incubating the slides in formic acid (70% distilled water) for 10 minutes, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (DAKO, Glostrup, Denmark, S2023). Sections were incubated overnight with biotinylated 4G8 antibody (Biolegend; San Diego, CA) diluted 1 / 2000 in antibody diluent containing background reduction components (DAKO, S3022). After extensive washing, streptavidin-HRP (PK6100 Elite, Vector Labs) was applied to the slides for 30 minutes, followed by color development with 3,3-diaminobenzidine (DAB) (DAKO, K3468). Slides were counterstained with hematoxylin, dehydrated, and permanently mounted with Vectamount (H-5000, Vector Labs). Images (20x) were generated using a NanoZoomer slide scanner (Hamamatsu Photonics) and analyzed using Matlab / Phaedra. Regions of interest (ROIs) were manually delineated according to the Franklin and Paxinos atlas (Franklin KB, Paxinos G. Mouse brain in stereotaxic coordinates. Waltham: Academic Press; 1997), and the percentage of DAB-labeled area to total area was calculated for each ROI.

[0364] To assess the effect on microhemorrhages, Perls staining was performed. Briefly, paraffin-embedded tissue sections were treated with an acidic ferrocyanide solution according to the following protocol. The iron ions (Fe3+) present in the microhemorrhages will bind to the ferrocyanide, resulting in the formation of a blue pigment known as Prussian blue. After dewaxing and rehydration, the sections were incubated in a 1 / 1 mixture of 2% potassium ferrocyanide (Sigma-Aldrich) and 2% glacial hydrochloric acid (Sigma-Aldrich) for 30 minutes. After rinsing the slides three times in distilled water, they were counterstained with nuclear fast red (Sigma-Aldrich), then rinsed in distilled water, dehydrated, and mounted (Vectamount, Vector Labs). Imaging was performed using a NanoZoomer slide scanner (Hamamatsu Photonics). The number of Perl-positive cells near the meninges was manually counted.

[0365] Results: In summary, minor microbleeds were observed in the baseline, isotype control, and BAMB31 treated groups ( Figure 8Biochemical analysis confirmed that BAMB31_2a reduced Aβ1-x concentration in the hippocampus by 34% (p<0.0001) relative to the isotype control antibody ( Figure 9 Furthermore, immunohistochemistry using antibody 4G8 showed a 23% (p<0.0001) and 37% (p<0.001) reduction in the hippocampus and cortex, respectively, compared to an isotype control antibody.

[0366] In conclusion, the efficacy of chronic intraperitoneal BAMB31 in reducing amyloid burden in a mouse model of plaque deposition was demonstrated without leading to an increased incidence of microhemorrhages, suggesting a favorable ratio of efficacy to toxicity following treatment with the BAMB31 antibody.

[0367] Example 10: Pharmacokinetics of BAMB31 HFA mAb

[0368] The pharmacokinetics of BAMB31 HFA mAb as wild-type IgG1 (BAMB674) and +YTE IgG1 (BAMB675) isoforms were evaluated in cynomolgus monkeys to directly compare properties in the peripheral circulation and brain.

[0369] Materials and Methods: Three animals per group received an intravenous (iv) bolus dose of 25 mg / kg of each mAb, and serum samples were collected over a 5-week period. Three additional monkeys received an i.v. bolus dose of 25 mg / kg of each mAb at week 6, and brain tissue was collected from each group on days 7 and 42 post-administration. In total, brain samples were collected from three cynomolgus monkeys on days 7 and 42 for each molecule.

[0370] Drug exposure analysis of BAMB674 and BAMB675 from in vivo cynomolgus monkey serum and brain tissue samples was performed using an electrochemiluminescence immunoassay (ECLIA) method that was individually tailored for the purpose, with endpoint determinations performed on a Meso Scale Discovery (MSD) Sector Imager S600. One assay format was applied to each compound and matrix, resulting in four independent methods for measuring exposure. The format is described as follows: mAb compounds were captured and detected using anti-human Fc specific (CH2 domain) mouse mAb. For brain tissue preparation, homogenates were prepared by freezing and crushing quick-frozen tissue and diluting it in buffer. The protein concentration of the tissue was verified and normalized using the BCA assay to produce the final protein concentration for the method. Raw data regression was performed using a 5-parameter logistic (automatic estimation) fit and 1 / Y2 standard curve weighting in Watson LIMS software.

[0371] A dual compartment (central (V)) with intravenous (iv) administration (dosing) to the central compartment, intercompartmental clearance (Q) and linear clearance (CL) was used. C) and organization (V T A ) compartmental pharmacokinetic (PK) model was developed to characterize the PK of BAMB674 and BAMB675 in cynomolgus monkeys. Figure 10 A schematic diagram of the model is shown.

[0372] Results: The terminal half-life of each antibody was calculated and is shown together with the cynomolgus monkey data and the 2-compartment model fit. Figure 11 The +YTE IgG1 isotype (BAMB675) exhibited an approximately 1.6-fold increase in half-life compared to the wild-type IgG1 isotype (BAMB674). The +YTE isotype (BAMB675) exhibited an approximately 3-fold increase in FcRn affinity compared to the wild-type IgG1 mAb (BAMB674), as shown in Table 5.

[0373] Levels in brain lysates were similar across regions and mAbs at day 7, but YTE mAb was consistently detected in only the brain regions and animals tested at day 42. Figure 12 This is consistent with increased exposure of the +YTE mAb at later time points.

[0374] In describing the present invention and its various embodiments, specific terminology is employed for the sake of clarity. However, the present invention is not intended to be limited to the specific terminology so selected. Those skilled in the relevant art will recognize that other equivalent components may be employed and other methods may be developed without departing from the broad concept of the present invention. All references cited anywhere in this specification are incorporated by reference as if each reference had been individually incorporated.

[0375] This application also provides the following solutions.

[0376] 1. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3 having the following polypeptide sequences:

[0377] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0378] b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively;

[0379] c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively;

[0380] d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively;

[0381] e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively;

[0382] f. SEQ ID NOs: 56, 57, 3, 4, 5 and 6, respectively;

[0383] g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively;

[0384] h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively;

[0385] i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively;

[0386] j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively;

[0387] k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively;

[0388] l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or

[0389] m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively;

[0390] The antibody or antigen-binding fragment thereof specifically binds to 3pE Ab, preferably human 3pE Ab.

[0391] 2. The isolated monoclonal antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 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 NO: 10, 12, 14, 18, 22, 53, or 55.

[0392] 3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising:

[0393] a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 22;

[0394] 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;

[0395] 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;

[0396] d. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 13 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 14;

[0397] e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 14;

[0398] f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 16 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 14;

[0399] g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 20 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 14;

[0400] 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;

[0401] 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;

[0402] 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

[0403] k. 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.

[0404] 4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of schemes 1 to 3, wherein the antibody or antigen-binding fragment thereof is chimeric.

[0405] 5. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of schemes 1 to 4, wherein the antibody or antigen-binding fragment thereof is human or humanized.

[0406] 6. An isolated monoclonal antibody comprising:

[0407] a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0408] b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0409] c. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 52; or

[0410] d. A heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 54.

[0411] 7. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 6.

[0412] 8. A vector comprising the isolated nucleic acid according to scheme 7.

[0413] 9. A host cell comprising the vector according to scheme 8.

[0414] 10. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 6 and a pharmaceutically acceptable carrier.

[0415] 11. A method for treating a condition associated with the formation of plaques containing β-amyloid protein in a subject in need thereof, the method comprising administering to the subject in need thereof the monoclonal antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 6, or the pharmaceutical composition according to Scheme 10.

[0416] 12. The method of claim 11, wherein the disorder is Alzheimer's disease.

[0417] 13. The method of 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 amyloidosis (HCHWA-D).

[0418] 14. A method of reducing plaques associated with Alzheimer's disease in a subject in need thereof, the method comprising administering to the subject in need thereof the monoclonal antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 6, or the pharmaceutical composition according to Scheme 10.

[0419] 15. A method for preventing the vaccination activity of 3pE Aβ in a subject in need thereof, comprising administering to the subject in need thereof the monoclonal antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 6 or the pharmaceutical composition according to Scheme 10.

[0420] 16. A method of producing the monoclonal antibody or antigen-binding fragment thereof according to any one of schemes 1 to 6, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions in which the monoclonal antibody or antigen-binding fragment thereof is produced, and recovering the antibody or antigen-binding fragment thereof.

[0421] 17. A method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 6, the method comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

Claims

1. A method for detecting amyloid-β peptide with pyroglutamate at the third residue (3pE Aβ) or a fragment thereof in a biological sample, the method comprising: (i) contacting the biological sample with an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to 3pE Aβ or a fragment thereof, and (ii) determining whether an immune complex is formed between the isolated monoclonal antibody or antigen-binding fragment thereof and 3pE Aβ or a fragment thereof; The isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3 and a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3 having the following polypeptide sequences: a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively; b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively; c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively; d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively; e. SEQ ID NOs: 56, 57, 3, 8, 5, and 6, respectively; f. SEQ ID NOs: 56, 57, 3, 4, 5 and 6, respectively; g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively; h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively; i. SEQ ID NOs: 1, 57, 3, 8, 5, and 6, respectively; j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively; k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively; l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively.

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

3. The method of claim 1 or 2, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 21 and a light chain variable region having a 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 a polypeptide sequence of SEQ ID NO: 13 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 14; e. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 14; f. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 16 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 14; g. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 20 and a light chain variable region having a 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. 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 method of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is chimeric.

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

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

7. The method of any one of claims 1 to 6, wherein the isolated monoclonal antibody comprises: a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38; b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38; c. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 52; d. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 52; e. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 54; or f. A heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO:

54.

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 the group consisting of a cerebrospinal fluid sample, a blood sample, a plasma sample, a serum sample, and a urine sample.

11. The method of any one of claims 1-10, wherein the biological sample is from a subject suffering from a disorder associated with the formation of plaques comprising beta-amyloid.

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

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

14. The method of any one of claims 1-13, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises a detectable label, and the method further comprises detecting the detectable label.

15. The method of claim 14, wherein the detectable label is selected from the group consisting of a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance.

16. The method of any one of claims 1-15, wherein the isolated monoclonal antibody or antigen-binding fragment thereof is immobilized on a solid substrate.

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