Antibodies to pyroglutamate amyloid-β and uses thereof

Abstract

The present invention provides antibodies or antigen-binding fragments thereof that bind to 3pE Aβ and methods for preparing and using the antibodies or antigen-binding fragments thereof, including uses in formulations, administration, and kits. The disclosed antibodies and antigen-binding fragments thereof and methods can be used for the diagnosis, prognosis, and treatment of Alzheimer's disease or other β-amyloid-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of antibodies to amyloid-β (Aβ) peptides and therapeutic methods using the same. In particular, the antibodies can be used to identify and treat amyloid-related diseases.

[0002] Reference sequence listing submitted electronically

[0003] This application contains a sequence listing, which is submitted electronically via EFS-Web as a sequence listing in ASCII format, with a file name of "JAB7013USPSP Sequence Listing" and a creation date of March 11, 2019, and a size of 76kb. The sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0004] Alzheimer's disease (AD) is a degenerative brain disorder characterized by progressive loss of memory, cognition, reasoning, judgment and emotional stability, which gradually leads to extreme mental decline and ultimate 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 the disease currently affects more than about five million people in the United States alone. It is currently incurable, and any treatment does not effectively prevent AD or reverse its symptoms or processes.

[0005] The brains of individuals with AD show 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 Dutch amyloidosis (HCHWA-D).

[0006] The major components of amyloid plaques are various amyloid-β (Aβ) peptides, which are generated by the cleavage of β-amyloid precursor protein (APP). It is assumed 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 cause early-onset AD in families provides strong evidence that altered amyloid metabolism is a central event in the pathogenic process that underlies the disease.

[0007] 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). The detectable amount of 3pE Aβ in CSF or plasma has not been reported, thus 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

[0008] 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 of 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 present 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 present invention preferentially bind to Aβ peptides containing 3pE compared to Aβ peptides that do not contain 3pE.

[0009] 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 sequence:

[0010] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0011] b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively;

[0012] c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively;

[0013] d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively;

[0014] e. SEQ ID NOs: 56, 57, 3, 8, 5 and 6, respectively;

[0015] f. SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively;

[0016] g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively;

[0017] h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively;

[0018] i. SEQ ID NO: 1, 57, 3, 8, 5 and 6 respectively;

[0019] j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively;

[0020] k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively;

[0021] l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or

[0022] m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively;

[0023] The antibody or antigen-binding fragment thereof specifically binds to 3pE Aβ, preferably human 3pE Aβ.

[0024] 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.

[0025] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0026] 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;

[0027] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 10;

[0028] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 11 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 12;

[0029] 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;

[0030] 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;

[0031] 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;

[0032] 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;

[0033] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 17 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 18;

[0034] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 19 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 18;

[0035] j. 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: 53; or

[0036] 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.

[0037] 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.

[0038] In certain embodiments, the isolated monoclonal antibody comprises:

[0039] a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0040] b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0041] 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

[0042] d. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:54.

[0043] 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 peptides (e.g., Aβ3pE-40 and Aβ3pE-42) with little or no cross-reactivity to other Aβ peptides or β-amyloid precursor protein (APP).

[0044] Also provided are isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof disclosed herein.

[0045] Also provided are vectors comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.

[0046] Also provided is a host cell comprising a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof of the present invention. Also provided is a hybridoma producing the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0047] 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.

[0048] Also provided is a method 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).

[0049] Also provided is a method of reducing plaques associated with Alzheimer's disease 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.

[0050] Also provided is a method of 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.

[0051] Also provided is a method for producing the monoclonal antibody or antigen-binding fragment thereof of the present invention, the method 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.

[0052] A method for producing the pharmaceutical composition of the present invention is also provided, 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.

[0053] One embodiment includes kits and devices comprising the above-described antibodies or antigen-binding fragments thereof.

[0054] 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

[0055] Figure 1 is a sensorgram (single cycle kinetics) of surface plasmon resonance label-free detection of affinity binding interaction of BAMB31_2a (mIgG2a) with human Aβ (3pE-40) peptide. The grey trace represents double reference subtracted data, while the black trace represents fitted values.

[0056] Figure 2 is a sensorgram (single cycle kinetics) of surface plasmon resonance label-free detection of affinity binding interaction of mE8c mIgG2a with human Aβ (3pE-40) peptide. The grey trace represents double reference subtracted data, while the black trace represents the fitted value.

[0057] FIG. 3A to FIG. 3I The reactivity of BAMB674 and BAMB675 to plaques by immunohistochemistry analysis in formalin-fixed, paraffin-embedded (FFPE) transgenic mouse brain tissue is shown. Results for primary antibody concentration 0.05 μg / mL are shown. Arrows indicate plaque-labeled areas for BAMB674 and BAMB675. (A) BAMB674; (B) BAM675; (C) Antibody I; (D) Antibody II; (E) B12L; (F) CI-C7; (G) hE8L; (H) R17L; (I) R17.

[0058] FIG. 4A to FIG. 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.

[0059] FIG. 5A to FIG. 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 stained brain sections and magnified areas.

[0060] FIG. 6A to FIG. 6DShown 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.

[0061] Figure 7 Shown is a graph demonstrating serum antibody concentrations at different time points following a single 20 mg / kg intraperitoneal (ip) administration in transgenic mice.

[0062] Figure 8 Shown is a graph demonstrating microhemorrhages after long-term treatment with isotype control and BAMB31_2a (mIgG2a) antibodies in PDAPP mice by evaluating the number of Perls-positive cells.

[0063] Fig. 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 is shown. Grey values ​​represent data points below the detection limit of the assay.

[0064] Fig.10 Schematic representation of the two-compartment model for BAMB674 and BAMB675 monkey PK characterization is shown.

[0065] Fig.11 Figures demonstrating PK data and observed 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 after intravenous (iv) bolus administration of 25 mg / kg in cynomolgus monkeys. Anti-Aβ3pE antibody (3pE-AB) μg / ml concentrations are displayed on the Y axis in logarithmic scales with the time days on the X axis. The calculated half-life (t1 / 2) of each mAb is displayed in the inset text.

[0066] Fig.12 A graph illustrating the brain concentrations observed for BAMB674 and BAMB675 is shown. Brain lysate levels at day 7 and day 42 of BAMB31 HFA mAb as wild type IgG1 (WT brain) vs. +YTE IgG1 (YTE brain) isotype following iv bolus administration of 25 mg / kg in cynomolgus monkeys. Anti-Aβ3pE antibody (3pE-AB) μg / ml concentrations are shown on the Y axis in a logarithmic scale versus time days on the X axis. DETAILED DESCRIPTION

[0067] 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, etc. included in this specification is intended to provide 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.

[0068] It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, and that the methods, reagents, compounds, compositions, or biological systems may vary. It should be understood in addition that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0069] 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.

[0070] 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.

[0071] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0072] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, should be understood to mean including the specified integer or group of integers but not excluding any other integer or group 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 these compositions, mixtures, processes, methods, articles, or apparatuses. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0073] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single 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".

[0074] As used herein, the term "consisting of" used throughout the specification and claims means including any listed integer or group of integers, but additional integers or groups of integers cannot be added to the specified method, structure, or composition.

[0075] As used herein, the term "consisting essentially of" 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.

[0076] Antibody

[0077] The present invention provides antibodies or antigen-binding fragments thereof that bind to 3pEβ peptides, in particular antibodies or antigen-binding fragments thereof that are preferentially present over 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 peptides, and methods for preparing hybridomas that produce antibodies or antigen-binding fragments thereof that bind to 3pE Aβ peptides. 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 containing antibodies or antigen-binding fragments thereof for use in the methods described herein.

[0078] According to a specific aspect, the present invention relates to 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 sequence:

[0079] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0080] b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively;

[0081] c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively;

[0082] d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively;

[0083] e. SEQ ID NOs: 56, 57, 3, 8, 5 and 6, respectively;

[0084] f. SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively;

[0085] g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively;

[0086] h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively;

[0087] i. SEQ ID NO: 1, 57, 3, 8, 5 and 6 respectively;

[0088] j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively;

[0089] k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively;

[0090] l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or

[0091] m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively;

[0092] The antibody or antigen-binding fragment thereof specifically binds to 3pE Aβ, preferably human 3pE Aβ.

[0093] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment comprising 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.

[0094] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising:

[0095] 1. 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;

[0096] m. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 10;

[0097] n. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 11 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 12;

[0098] o. 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;

[0099] p. 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;

[0100] q. 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;

[0101] r. 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;

[0102] s. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 17 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 18;

[0103] t. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 19 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 18;

[0104] u. 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: 53; or

[0105] v. 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.

[0106] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 a polypeptide sequence of SEQ ID NO: 21; and a light chain variable region having a polypeptide sequence of SEQ ID NO: 22 or 53 or 55.

[0107] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 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 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.

[0108] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 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 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.

[0109] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 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 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.

[0110] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 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 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.

[0111] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 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 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.

[0112] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 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 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.

[0113] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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 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 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.

[0114] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising 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.

[0115] In another specific aspect, the isolated monoclonal antibody comprises:

[0116] a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0117] b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0118] 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

[0119] d. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:55.

[0120] 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.

[0121] 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.

[0122] 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 its antigen binding fragment 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).

[0123] In another general aspect, the present invention relates to isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof of the present invention. It will be appreciated by those skilled in the art that the coding sequence of a protein may be altered (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, it will be appreciated by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibodies or antigen-binding fragments thereof of the present invention may be altered without changing the amino acid sequence of the protein.

[0124] In another general aspect, the present invention relates to a vector comprising a nucleic acid encoding the separation of the 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 establishing 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 acids 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.

[0125] In another general aspect, the present invention relates to a host cell comprising a nucleic acid encoding the separation of the monoclonal antibody of the present invention or its antigen-binding fragment. In view of the present disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the antibody of the present invention or its antigen-binding fragment. In some embodiments, the host cell is Escherichia coli TG1 or BL21 cells (for expressing, for example, scFv or Fab antibodies), CHO-DG44 or CHO-K1 cells or HEK293 cells (for expressing, for example, full-length IgG antibodies). 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.

[0126] 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 a monoclonal antibody or antigen-binding fragment thereof under conditions that produce a 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.

[0127] The present invention provides an isolated antibody or antigen-binding fragment thereof that binds to 3pE Aβ. The term "antibody" herein refers to an immunoglobulin that is capable of binding to an antigen or a portion thereof, in particular an immunoglobulin that is 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.

[0128] The antigen-binding fragment of an antibody refers to a fragment of an antibody that can bind to an antigen to which the whole antibody binds, and competes with the whole antibody for antigen binding. The antigen-binding fragment comprises a portion of the whole antibody that allows antigen binding (i.e., the variable region of the whole antibody). The antigen-binding fragment may include, but is not limited to, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (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 fragment that binds to an antigen but does not contain the structure of a whole antibody.

[0129] 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 L). The 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).

[0130] 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 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); Scaviner et al., Exp. Clin. Immunogenet. 16:234-240 (1999); Lefranc et al., Nucleic Acids Res. 43:D413-422 (2015)).

[0131] When used in the context of antibodies, "isolated" means changed "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). Antibodies may be present in compositions such as immunoassay reagents that are not naturally occurring compositions, and therein are still isolated antibodies within the meaning of the term (as used herein).

[0132] The method for producing antibodies comprises inoculating the host with the desired immunogen. 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 capable of establishing a mature immune response. Immunity procedures are well established in the art and are described in many papers and publications, including "The Immunoassay Handbook" (2nd edition, David Wild, ed., Nature Publishing Group, 2000).

[0133] Preferably, the immunogen comprising the features of the 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 dipladen mycolate (MPL-TDM).

[0134] 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 performed 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.

[0135] 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 from a host, harvesting the monoclonal antibodies that secrete or have the potential to secrete lymphocytes, fusing the lymphocytes to immortalized cells, and selecting cells that secrete the desired monoclonal antibodies.

[0136] 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 required, peripheral blood lymphocytes can be used, but spleen cells or lymphocytes from other mammalian sources are preferred.

[0137] Lymphocytes can be fused with immortalized cell lines to form hybridoma cells, which can be promoted by using a fusion agent, such as a process promoted by polyethylene glycol. By way of example, mutant rodents, cattle or human myeloma cells that are immortalized by transformation can be used. Relative to unfused immortalized cells, substantially pure fusion tumor cell groups are preferred. 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 phosphoribosyl transferase (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.

[0138] 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 following fusion. Preferred immortalized cell lines include myeloma cell lines purchased from the American Type Culture Collection (Manassas, VA).

[0139] 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 include: (i) selecting a host for antibody production; (ii) inoculating the host with a desired immunogen; (iii) fusing the cell line of the inoculated host with a continuously dividing cell 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.

[0140] The methods of the present invention include methods for producing hybridoma cell lines capable of producing monoclonal antibodies that bind to 3pE Aβ peptides. Hybridomas can be prepared by the following method: an animal (such as a Balb / c mouse) from which hybridomas can be produced is immunized with an initial intraperitoneal injection of a desired immunogen (such as an 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 commonly known to those of ordinary skill in the art, preferably using SP2 / 0 cells, by 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 to 3pE Aβ peptides. Screening can be performed in standard assays such as ELISA or RIA assays. One aspect of the present invention is a method for producing a hybridoma cell line that produces monoclonal antibody BAMB31_1 or a humanized form thereof.

[0141] Monoclonal antibodies can also be produced by recombinant methods known in the art, for example, as described in U.S. Pat. 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 to probe DNA isolated from a monoclonal antibody hybridoma cell line that secretes Aβ-specific antibodies with pyroglutamate.

[0142] 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 digesting antibody molecules with pepsin; and Fab fragments, which can be generated by reducing the disulfide bonds of the F(ab')2 fragments. Alternatively, a Fab expression library 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. Pat. Nos. 5,761,894 and 5,587,458). Fv and sFv fragments are the only species with complete combining sites lacking constant regions; therefore, they may show reduced nonspecific binding. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Pat. No. 5,642,870.

[0143] Therefore, an object of the present invention is to provide an isolated monoclonal antibody expressed by the above hybridoma cells, which antibody can specifically recognize 3pE Aβ. The isolated monoclonal antibody can be expressed by hybridoma cells or recombinantly.

[0144] 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 or APP that do not contain 3pE.

[0145] Table 1 provides the amino acid sequences of the antibodies of the invention. The CDRs of the heavy chain variable region and the light chain variable region as defined by Kabat, Chothia and IMGT are shown in separate sequences.

[0146] Table 1: 3pE Aβ monoclonal antibody sequences

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] The terms "same" 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.

[0161] For sequence comparison, usually one sequence is used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and program parameters for the sequence algorithm are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage for the test sequence relative to the reference sequence based on the specified program parameters.

[0162] 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 the similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA, 85:2444 (1988). 85:2444 (1988)), by algorithms (GAP, BESTFIT, FASTA and TFASTA, computerized implementations of 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, Inc. (1995 supplement) (Ausubel)).

[0163] 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) Journal of Molecular Biology, Vol. 215, pp. 403-410 (J. Mol. Biol. 215: 403-410) and Altschul et al. (1997) Nucleic Acids Res. Vol. 25: 3389-3402 (Nucleic Acids Res. 25: 3389-3402), respectively. Res. Vol. 25: pp. 3389-3402. Software for performing BLAST analysis 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 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 containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased.

[0164] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatched residues; always<0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits 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 score 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 value (E) of 10, M=5, N=-4, and a comparison of two chains by default. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation value (E) of 10, and a BLOSUM62 scoring matrix by default (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0165] In addition to calculating the percentage of 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 similarity measure 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 a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0166] 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.

[0167] In vitro methods

[0168] 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 invention include, but are not limited to, competitive (reagent-limited) assays in which a labeled analyte (analyte analog) in a sample and the analyte competes for the antibody, as well as single-site immunoassays in which the antibody is labeled; etc.

[0169] 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 cultures 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, etc., and as is also well known, they all rely on the formation of antigen-antibody immune complexes, wherein for the purpose of the assay, the antibody or its antigen-binding fragment can be detectably labeled with, for example, a radioactive, enzymatic, luminescent or fluorescent marker, or the antibody or its antigen-binding fragment can be fixed on an insoluble carrier. Therefore, the object of the present invention is to provide an immunoassay for determining or detecting Aβ3pE or its fragments in a sample, the method comprising contacting the sample with an antibody or its antigen-binding fragment against Aβ3pE or its fragment according to the present invention, and determining whether an immune complex is formed between the antibody or its antigen-binding fragment and Aβ3pE or its fragment. 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 (particularly the amount of Aβ3pE or a fragment thereof) in the solution to be tested 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, competition, immunoassay and sandwich methods are suitable. In terms of sensitivity and specificity, the sandwich method is particularly preferred.

[0170] 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, whereby the amount of Aβ3pE or its fragment in the test solution can be determined. The first reaction and the second reaction can be performed simultaneously or sequentially.

[0171] In the measuring 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 usually made detectable by conjugation to an appropriate substrate which 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, avidin-biotin systems can also be used to label 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 fixation 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.

[0172] 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 included and contacted with an appropriate amount of a first antibody to produce an immune complex. The 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 to 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 a complex can be easily detected using techniques known in the art, and the complex consists of antigens 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 changed Aβ3pE or its fragment can be determined.

[0173] In vivo methods

[0174] Aspects of the invention relate to methods for preventing, ameliorating, treating and / or reducing amyloid-β deposition in amyloid-β-related disorders, comprising administering an antibody or antigen-binding fragment thereof disclosed herein to a subject of the invention in a therapeutically effective amount. Additional aspects of the invention include pharmaceutical compositions for preventing, ameliorating, treating and / or reducing amyloid deposition in amyloid-β-related disorders, comprising an antibody or antigen-binding fragment thereof as disclosed herein. The methods of the invention include administering an effective amount of one or more antibodies or antigen-binding fragments thereof as described herein to a subject in need thereof.

[0175] 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 the human body, the method comprising administering a therapeutically effective amount or a prophylactically effective amount of an antibody according to the present invention or an immunologically reactive fragment thereof, preferably peripherally, to a person in need of such treatment, the antibody specifically binding to human Aβ3pE. On the other hand, the present invention relates to a method for inhibiting the formation of amyloid plaques and / or a method for clearing amyloid plaques in humans, the method comprising administering an effective amount of an antibody according to the present invention to a human subject in need of such inhibition or clearance, wherein the antibody chelates 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.

[0176] A subject in need thereof is a person 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 Dutch amyloidosis (HCHWA-D).

[0177] Humanized antibody is an antibody from a non-human species, and its protein sequence has been modified to increase the similarity of the naturally occurring antibody variants with them and people. Generally speaking, the protein sequence of humanized antibody is substantially the same as that of human variant, except that its complementary determining region (CDR) fragment is responsible for some or all of the non-human sources of the ability of antibody binding to its target antigen. The framework region of the variable region is replaced by the corresponding human framework region, retaining the non-human CDR substantially intact. In some cases, humanized antibody does have a small amount of substitution in one or more of the non-human CDR regions, to retain the binding affinity and / or the dissociation constant of the non-human antibody.

[0178] 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 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.

[0179] 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 antibodies 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.

[0180] In the method for treating and preventing plaques characterized by the formation of amyloid-beta, the antibodies or antigen-binding fragments thereof (including immunologically active fragments) of the present invention are administered to subjects 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 antibodies or their binding fragments can be directly administered to the ventricular system, cerebrospinal fluid or brain parenchyma, and the techniques for accessing these locations are 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 antibodies or their binding fragments of the present invention are effective. The advantages of the present invention include the ability of antibodies or their antigen-binding fragments to exert their beneficial effects even if they are not directly provided to the central nervous system itself.

[0181] Pharmaceutical compositions for administration are designed to be suitable for 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.

[0182] 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.

[0183] Peripheral systemic delivery by intravenous injection, intraperitoneal injection or subcutaneous injection is preferred. Suitable carriers for such injections are very simple. However, in addition, administration can also be performed by means of nasal aerosols or suppositories through the mucous membrane. Formulations suitable for such modes of administration are well known and generally include surfactants that are conducive to transmembrane transfer. Such surfactants are generally derived from steroids or cationic lipids, such as N-[1-(2,3-dioleyl)propyl-N,N,N-trimethylammonium chloride (DOTMA) or various compounds, such as cholesterol hemisuccinate, phosphatidylglycerol, etc.

[0184] According to selected specific mode of administration, mainly based on fluid volume, viscosity etc., select the concentration of humanized antibody in the preparation as low as about 0.1 % by weight up to about 15 % by weight or 20 % by weight.Therefore, the typical pharmaceutical composition for injection can be made into the sterile buffered water comprising 1mL phosphate buffered saline and 1mg to 100mg humanized antibody of the present invention.Preparation can be aseptically filtered after preparing preparation, or in other words, made into microbiologically acceptable.The typical composition for intravenous infusion can have the volume of up to 250mL fluid such as sterile Ringer's solution and 1mg / mL to 100mg / mL or higher antibody concentration.

[0185] 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 an embodiment, 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 may 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 dosage range of about 0.3 mg / kg to about 60 mg / kg. In an exemplary treatment regimen, the antibody is administered intraperitoneally in a dosage range 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.

[0186] As used herein, the term "about" when referring to a measurable value such as an amount is meant to encompass the following variations between the specified value: ±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.

[0187] Exemplary treatment regimens require administration once every two weeks or once a 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 may be weekly, monthly, or annual. Intervals may also be irregular, as indicated by measuring the blood levels of antibodies to A β in the subject. Alternatively, antibodies may be administered as sustained-release formulations, 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.

[0188] The dosage and frequency of administration may vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals may be required until the progression of the disease slows or terminates, and preferably until the subject exhibits partial or complete improvement in symptoms of the disease. Then, a preventive mechanism may be administered.

[0189] In some methods, the dosage 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. Dosage and frequency vary depending on the half-life of the antibody in the subject.

[0190] 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 combined treatment regimen in which one or more additional therapeutic agents are also used to treat the individual.

[0191] When used for in vivo therapy, the antibody or its antigen-binding fragment of the present invention is administered to an individual in a therapeutically effective amount, such as to reduce, remove or prevent β-amyloid plaques or improve the amount of cognitive function of a subject suffering from AD or other β-amyloid-related diseases. The antibody or its antigen-binding fragment is administered to an individual by intramuscular, intraperitoneal, cerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes according to known methods, such as intravenous administration, for example, bolus or continuous infusion over a period of time. 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 disorders in which amyloid deposition occurs in the brain, the antibody or its antigen-binding fragment of the present invention may be administered in combination with other medicaments that enhance the medicament of the present invention to pass through the blood-brain barrier.

[0192] In one embodiment of the invention, the antibodies or antigen-binding fragments thereof of the 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. Induction of plaque removal can be performed 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 invention can inhibit the plaque seeding activity of 3pE Aβ. The possible enrichment of 3pE Aβ in plaques compared to vascular amyloid can increase the therapeutic safety window of immunotherapy.

[0193] Kits and Devices

[0194] The present invention provides kits and devices that can be used for the above methods. Preferably, the kits and devices contain antibodies or antigen-binding fragments thereof that bind to 3pE Aβ. In addition, the kit may contain reagents and instruction materials. The instructions may be, for example, printed on paper and / or provided in an electronically readable medium. Alternatively, the instructions may be provided by directing the user to an Internet website, such as an Internet website specified by the manufacturer or distributor of the kit.

[0195] The reagents contained in the kit of the present invention may be provided in various forms of containers so that the activities of the various components are substantially maintained while the components themselves are not substantially adsorbed or altered by the material of the container.

[0196] 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.

[0197] Implementation

[0198] Embodiment 1 is 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 sequence:

[0199] a. SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively;

[0200] b. SEQ ID NO: 1, 7, 3, 4, 5 and 6 respectively;

[0201] c. SEQ ID NOs: 1, 7, 3, 8, 5 and 6, respectively;

[0202] d. SEQ ID NO: 1, 2, 3, 8, 5 and 6, respectively;

[0203] e. SEQ ID NOs: 56, 57, 3, 8, 5 and 6, respectively;

[0204] f. SEQ ID NO: 56, 57, 3, 4, 5 and 6, respectively;

[0205] g. SEQ ID NOs: 56, 58, 3, 4, 5 and 6, respectively;

[0206] h. SEQ ID NOs: 56, 7, 3, 8, 5 and 6, respectively;

[0207] i. SEQ ID NO: 1, 57, 3, 8, 5 and 6 respectively;

[0208] j. SEQ ID NOs: 56, 7, 3, 4, 5 and 6, respectively;

[0209] k. SEQ ID NOs: 1, 57, 3, 4, 5 and 6, respectively;

[0210] l. SEQ ID NO: 1, 58, 3, 4, 5 and 6 respectively; or

[0211] m. SEQ ID NO: 56, 2, 3, 4, 5 and 6, respectively;

[0212] The antibody or antigen-binding fragment thereof specifically binds to 3pE Aβ, preferably human 3pE Aβ.

[0213] 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.

[0214] Embodiment 3 is an isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, comprising:

[0215] 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;

[0216] b. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 10;

[0217] c. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 11 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 12;

[0218] 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;

[0219] 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;

[0220] 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;

[0221] 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;

[0222] h. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 17 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 18;

[0223] i. a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 19 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 18;

[0224] j. 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: 53; or

[0225] 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.

[0226] Embodiment 4 is an 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.

[0227] Embodiment 5 is an 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.

[0228] Embodiment 6 is an isolated monoclonal antibody comprising:

[0229] a. a heavy chain amino acid sequence comprising SEQ ID NO: 37 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0230] b. a heavy chain amino acid sequence comprising SEQ ID NO: 39 and a light chain amino acid sequence comprising SEQ ID NO: 38;

[0231] 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

[0232] d. A heavy chain amino acid sequence comprising SEQ ID NO:39 and a light chain amino acid sequence comprising SEQ ID NO:54.

[0233] 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.

[0234] Embodiment 8 is a vector comprising the isolated nucleic acid according to embodiment 7.

[0235] Embodiment 9 is a host cell comprising the vector according to embodiment 8.

[0236] 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.

[0237] Embodiment 11 is a method of treating a condition associated with the formation of plaques containing β-amyloid protein in a subject in need thereof, the method comprising administering to a 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.

[0238] Embodiment 12 is a method according to embodiment 11, wherein the disorder is Alzheimer's disease.

[0239] Embodiment 13 is a method according to embodiment 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).

[0240] Embodiment 14 is a method of reducing plaques associated with Alzheimer's disease in a subject in need thereof, the method comprising administering to a 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.

[0241] Embodiment 15 is a method of preventing vaccination activity of 3pE Aβ in a subject in need thereof, the method comprising administering to a 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.

[0242] 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.

[0243] 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.

[0244] Example

[0245] The invention can be further understood in view of the following non-limiting examples.

[0246] Example 1: Monoclonal Antibody Generation and Humanization Process

[0247] 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.

[0248] Hybridoma and antibody production: Mice showing the highest serum titers were selected for fusion, while spleens from other mice were isolated and frozen in liquid nitrogen. On day 4, all mice were boosted intraperitoneally with 100 μg H2N-pEFRHDSGC-COOH (SEQ ID NO: 47) coupled to BSA (Merck; Kenilworth, NJ) in saline before fusion or spleen extraction. Mouse spleen cells were fused with SP2 / 0 cells (ATCC; Manassas, VA) by a modified procedure of Kohler and Milstein (Euro. J. Immunol., 1976; 292-295). Hybridomas were seeded in 30×96-well plates and screened 10 days later in a direct ELISA against 0.5 μg / well unconjugated Aβ3pE-40 peptide (AnaSpec; Fremont, CA). Positive cells were tested for (lack of) cross-reactivity to 0.5 μg / ml coated Aβ1-40 peptide (AnaSpec) and immediately subcloned.

[0249] 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.

[0250] 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.

[0251] Direct ELISA for antibody selection: The screening ELISA for detecting the above-mentioned Aβ3pE-40 antibodies was a direct ELISA coated with 0.5 μg / ml free human Aβ3pE-40 peptide (SEQ ID NO: 40) in 50 μl / well coating buffer (10 mM Tris, 10 mM NaCl and 10 mM NaN3, pH 8.5) at 4°C overnight.

[0252] The next day, the plate was 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, the bound monoclonal antibody was detected at 37°C for 1 hour with 50 μl / well of sheep anti-mouse IgG conjugated with horseradish peroxidase (Amersham-Pharmacia Biotech; Little Chalfont, United Kingdom). Both reagents were diluted in 0.1% casein / PBS. The plate was washed and 50 μl of 0.42 mM 3,5,3',5'-tetramethylbenzidine (Biorad), 0.003% (volume / volume) H2O2 (Biorad) of 100 mM citric acid (Biorad; Hercules, CA), 100 mM disodium hydrogen phosphate (pH 4.3) (Biorad) were added as substrates. Reactions were allowed to proceed for a maximum of 15 min at room temperature on a plate shaker, after which color development was stopped with 50 μl / well of 2N H2SO4 and the plates were read at 450 nm on a microtiter plate reader (Thermomax, Molecular Devices). Selected monoclonal antibodies were tested for cross-reactivity with full-size human free Aβ1-40 using the same direct ELISA as the screening assay.

[0253] From 17 Aβ3pE-40 reactive clones, BAMB31_1 was selected based on affinity and selectivity for further characterization (see Examples 2 and 3). It was determined that this antibody had 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 with mouse IgG2a Fc, these isotypes have different activities and protein spectra. Compared with 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, which binds to mouse FcγRI, FcγRIII and FcγRIV receptors and mouse C1q, thereby having complement, antibody-dependent cellular toxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) activities that can contribute to the clearance of Aβ plaques.

[0254] The sequence of the BAMB31 heavy chain was changed from mouse IgG1 to mouse IgG2a to form BAMB31_2a. The retention of reactivity after V region cloning was confirmed by SPR method as described below.

[0255] Humanization process: The parent antibody BAMB31-2a (mIgG2a) was humanized using a program similar to that of Singh et al. (MAbs 2015; 7(4): 778-91), except that CDR-H2 was described according to the AbM definition (Martin, AC, PNAS 86: 9268-9277, 1989) used for this work. In brief, complementary determining regions (CDRs) were identified in the mouse parental sequence. These sequences were compared with human germline, and 4 human germline heavy chains and 2 human germline light chain frameworks in which mouse CDRs were transplanted were selected. The human J segments of the VL and VH of each parental antibody were selected by comparing mouse and human J segment sequences to maximize sequence identity. Molecular models of the Fv regions of parental mAbs were generated in MOE (CCG; Montreal, Canada) using default parameters. The resulting model was graphically examined 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 one chain is suitable for people's framework, and back mutation is determined based on antigen binding.Then humanized VH and VL are combined to obtain final candidate antibody.

[0256] Library clones were expressed as Fabs in E. coli and tested for binding to peptides by ELISA, and the signals were compared to the complete mouse parent molecule. Molecular signals showing binding greater than 80% of the mouse parent were selected for sequencing. The sequences were analyzed, and human-adapted heavy chains and human-adapted light chains were selected to be combined and expressed as monoclonal human IgG1 antibodies. For all VH / VL humanized pairs of each antibody, expression, purification and evaluation of antigen binding, Epivax computer simulation of immunogenicity risk, number of residues restored to mouse sequences and biophysical properties were performed.

[0257] Results: Some residues needed to be reverted to the mouse sequence to maintain parental binding. Retention of binding, Epivax computer simulation immunogenicity risk, and biophysical properties were not dependent on the number of reverted to mouse sequences, but rather on the positions reverted to mouse sequences. Representative HFA mAb characterization results from this analysis are shown in Tables 2 and 3.

[0258] Table 2: Example of BAMB31HFA characterization results

[0259]

[0260] BAMB246 parental human IgG1 chimera

[0261] Values ​​outside the expected range are indicated in bold: total number of residues restored to mouse sequence >5; Hc and Lc Epivax risk scores >-10; V region combined Epivax risk score >-20; SEC% monomer value <95% kd(1 / s) value >1.00E-04; KD(M) value >2.50E-11; Tstart(℃)<60; Tm1(℃)<65; Tagg(℃)<65.

[0262] Reduction of post-translational modification risk: The parent antibody and human framework adapted variants contain the NG post-translational deamidation modification motif in HCDR2. To address this potential issue, separate libraries were created for N residues and G residues using degenerate oligonucleotides. These generated new sequences randomly introduced all 20 amino acids for each position. Each library was screened for retained binding. Variant antibodies that showed similar binding to the mouse parent were selected for sequencing.

[0263] 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 the 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). It is known that these mutations increase the affinity to FcRn and extend the 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.

[0264] Table 3: Characterization of BAMB674 and BAMB675

[0265] Properties BAMB674 BAMB675 Isotype wt IgG1 +YTE IgG1 FWf IGHV1-46*03 IGHV1-46*03 Hc FW positions requiring mouse residues M48I、M70L M48I、M70L Hc CDR2 NG motif reduction N55S N55S FWf 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

[0266] Table 3a: Thermal stability

[0267]

[0268] * N = 1

[0269] Example 2: Thermal stability test

[0270] For variants adapted to the BAMB31 human framework, their thermal stability was assessed using nano differential scanning fluorimetry (NanoDSF) to measure melting onset temperature (Tstart), first melting transition temperature (Tm1) and initial aggregation detection temperature (Tagg). The data for some variants are shown in Table 2 (columns 6 to 8) and Table 3 (rows 10 to 12) and Table 3a.

[0271] Materials and methods: The thermal stability of the samples was determined using an automated Prometheus instrument. The measurements were performed by loading the samples from a 384-well sample plate into a 24-well capillary. Each sample was run in duplicate. 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 0.3mg / mL to 1mg / mL. The intrinsic fluorescence of the molecule at 330nm and 350nm 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 the back reflection technique 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 the lead candidate, which is often monitored as an indicator of long-term sample stability under changing conditions.

[0272] Example 3: Epivax in silico immunogenicity risk assessment

[0273] 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 continuous amino acid 9-mers to identify potential HLA class II binding sequences. The database includes the most common HLA types covering about 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 a T effector or T regulatory cell, 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.

[0274] Example 4: Analytical characterization of purified mAbs

[0275] 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 multi-channel spectrophotometer and calculated using the extinction coefficient based on the amino acid sequence.

[0276] SE HPLC of purified antibodies was performed in the following manner: on a TOSOH TSKgel BioAssist G3SWxl column, samples were 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.

[0277] Example 5: Binding kinetics and affinity measurements

[0278] Surface plasmon resonance (SPR) is a label-free detection method for studying biomolecular interactions. Monitoring minute 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 interaction 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) was studied.

[0279] Materials and methods

[0280] 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 salt 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.

[0281] Using Optical Biosensor T200 Affinity measurements were performed. Kinetic analysis was performed according to a 1:1 binding fit model using the Biacore T200 Evaluation Software (version 2.0).

[0282] 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, after which various concentrations of Aβ peptides or proteins (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 PBS containing 0.005% surfactant P20 (Tween TM20) in HEPES buffered saline. 30 μL of 10mM Gly pH 1.5 was injected twice at a rate of 100 μL / min to regenerate the surface. The reported data is the difference in SPR signals between the flow cell containing the captured antibody and the reference cell without the captured antibody. The additional instrumental contribution to the signal is removed by subtracting the data of the blank injection from the signal deducted from the reference. When applicable, the data is analyzed by fitting the association phase and dissociation phase (global fit) at all concentrations using the Biaevaluation software (Biacore, Inc.) with a 1:1 binding model. Otherwise, the data is qualitatively evaluated as yes / no binding.

[0283] 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 solution) for 10 minutes, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (DAKO; Glostrup, Denmark, S2023). The sections were incubated for 1 hour 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 (DAKO, S3022) containing background reduction components). After extensive washing, HRP-labeled 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 color development with 3,3-diaminobenzidine (DAB) (DAKO, K3468). The slides were counterstained with hematoxylin, dehydrated and permanently mounted with Vectamount (H-5000, VectorLabs).

[0284] 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) confirming 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. Equilibrium binding constant (affinity, K D ) and the kinetic rate constant (k a / k d ) are shown in Table 4.

[0285] Table 4: Kinetics of J&JPRD / Aβ / pE3 / 1, BAMB31 and mE8cmIgGa

[0286]

[0287] Kinetic analysis of BAMB31 HFA mAbs (BAMB674 and BAMB675) with reduced risk of Hc CDR2 NG deamidation showed retention of binding affinity to Aβ3pE-28 peptide (SEQ ID NO: 42) relative to BAMB31_2a (mIgG2a) parent and BAMB246 (human IgG1 chimera) parent. Equilibrium binding constant (affinity, K D ) and the kinetic rate constant (k a / k d ) are shown in Table 5.

[0288] 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.

[0289] Table 5: 3pE Aβ binding kinetics of BAMB31 HFA and mE8c HFA molecules

[0290]

[0291] Human chimeric antibodies have mouse variable regions in the human IgG1 constant region.

[0292] Table 5a: Binding affinity comparison

[0293] 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

[0294] HU-3pE-β-amyloid concentration range 0.6nM to 4.5nM

[0295] The higher affinity measured by SPR also translates 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 obtained visible plaque marking, but to varying degrees. At a concentration of 0.1 μg / mL, antibodies C1 to C7 and R17L did not produce visible plaque marking, according to the low affinity measured by SPR. At a concentration of 0.05 μg / mL, BAMB674 and BAMB675 showed plaque marking, while for the comparison molecules, there was no visual plaque marking at this concentration (Figure 3). At a concentration of 0.025 μg / mL, there was no visible plaque marking (almost completely) for all tested molecules.

[0296] In summary, the immunohistochemistry data confirm the SPR data demonstrating that BAMB674 and BAMB675 have higher affinity compared to the comparator molecules.

[0297] 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.

[0298] Table 6: Selective binding kinetics of BAMB246 (human IgG1 chimera) and HFA mAbs to relevant targets

[0299]

[0300] Scrambled 3pE-28 is a human Aβ3pE-28 peptide in which amino acids 3-9 are scrambled.

[0301] Fold selectivity was determined by dividing the KD in Table 4 by the KD in Table 3.

[0302] >1.20E-06K D (M) No antigen binding was detected at the highest concentration tested, 1.2 μM.

[0303] 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 translates into a longer circulating half-life for the +YTE antibody in cynomolgus monkey pharmacokinetic studies ( Fig.11 ).

[0304] Table 7: Comparison of FcRn binding affinity of HFA mAb as wild type and +YTE IgG1

[0305]

[0306] KD(M) values ​​are the average of 5 to 6 independent replicates.

[0307] Example 6: Sandwich ELISA for cross-reactivity testing

[0308] 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.

[0309] 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 monoclonal antibodies 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 with HRPO-labeled secondary antibodies (JRF / cAβ40 / 28-HRPO or JRF / cAβ42 / 26-HRPO) at 4°C. After overnight incubation, the plates were washed and the assay was developed with a TMB peroxide EIA substrate kit (Biorad) according to the manufacturer's recommendations.

[0310] 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 a concentration of up to 10 ng / mL (Figure 4).

[0311] Figure 7 : Immunohistochemistry for detection of antibody reactivity against plaques in transgenic mouse and human AD brain tissue study

[0312] Antibody reactivity to plaques was studied in formalin-fixed, paraffin-embedded (FFPE) and cryopreserved brain tissue.

[0313] Materials and methods

[0314] Formalin-fixed paraffin-embedded brain: 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). The 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-labeled 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).

[0315] Cryopreserved brain: Human brain samples were snap frozen, cryostat sliced ​​(20 μm thickness) and stored at -80°C before use. The slices 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 PBS 1x + 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 slices for 1 hour. After thorough washing, the slides were incubated with HRP-conjugated anti-mouse secondary antibodies (Envision, DAKO, K4000) followed by color development DAB labeling (DAKO, K3468). The 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).

[0316] Results: The reactivity of BAMB264 (human IgG1 chimera) and BAMB674 and BAMB675 was confirmed on FFPE tissues of transgenic mice. FIG. 5A to FIG. 5F ). In addition, BAMB31_2a (mIgG2a) demonstrated significant plaque labeling in cryopreserved AD brain tissue (Figure 6). In frozen sections of human brain, most plaques detected by antibody 4G8 were also labeled by BAMB31 ( FIG. 5A to FIG. 5F).++

[0317] Example 8: Serum antibody levels after administration to transgenic mice

[0318] Serum antibody levels after treatment with BAMB31 and the comparator molecule mE8c were investigated.

[0319] Materials and Methods: Aged 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). After antibody administration, whole blood was collected at intermediate time points (24 and 48 hours after injection) via the great saphenous vein and at 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 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 buffer + 0.05% Tween-20) and further diluted to 0.1 ng / ml in blocking buffer. After washing (PBS buffer + 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 TM The plates were incubated at room temperature for 1 hour. After the sample incubation, the plates were washed and incubated at room temperature for 1 hour with peroxidase-AffiniPure goat anti-mouse IgG (Fcγ subclass 2a specific) antibody. The plates were washed and developed by adding TMB peroxidase EIA substrate kit (1 step, Pierce) to the wells. Development was stopped by adding 2NH2SO4 to the wells after 2 minutes, and the plates were read at 450nm using an EnVision multi-mode plate reader (Perkin Elmer).

[0320] 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 isotype control antibodies, antibody concentrations gradually decreased over time (up to about 30% decrease on day 4), while a significantly higher decrease over time was observed for mE8c, with a decrease of about 97% on day 4 ( Figure 7 ).

[0321] In conclusion, differences in the pharmacokinetic profiles of BAMB31_2a and mE8c following intraperitoneal injection in a mouse model of plaque deposition were shown, indicating that clearance after treatment with the BAMB31_2a antibody was slower than that of mE8c.

[0322] Example 9: Chronic efficacy study in a transgenic mouse model

[0323] 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.

[0324] Materials and methods: PDAPP (V717F) transgenic mice (mean age of 18.3 months at the beginning of the study) were treated intraperitoneally weekly with BAMB31-2a antibodies (mIgG2a) at a dose of 30 mg / kg for 12 weeks. A control group receiving injections of mIgG2a isotype control antibodies was included in the experiment. Animals were euthanized on the 7th day after the last intraperitoneal injection (mean age of 21.1 months at the end of the study). Before collecting tissue, mice received PBS perfusion. 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.

[0325] To evaluate 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 in an inverted rotating wheel at room temperature for 3 hours. The resulting homogenate was stored at -80°C before analysis by MSD sandwich immunoassay.

[0326] 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, peptides were further diluted in 0.5M GuHCl+5mM Tris-HCl-pH 8.0 (extraction buffer was diluted 10-fold in PBS with 0.1% casein). GuHCl extracts were thawed and diluted 1:10 in ice-cold 0.1% casein in PBS and centrifuged at 20,000g for 20 minutes at 4°C. The supernatant was recovered for sandwich MSD assays, and the samples were further diluted in 0.5M GuHCl+5mM Tris-HCl-pH 8.0 (extraction buffer was diluted 10-fold in PBS with 0.1% casein).

[0327] 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. Standards and samples were incubated with biotinylated secondary antibodies overnight at 4°C. After overnight incubation, the plates were washed and incubated with secondary detection reagents (streptavidin-SULFO-TAG TM The plates were washed and 2X read buffer T was added before reading the plates according to the manufacturer's recommendations. 2 Aβ concentrations were determined from the standard curve of a four-parameter logistic model with a weighting function.

[0328] The combination JRF / AβN / 25+4G8-biotin antibody was used to study the Aβ1-x concentration in brain homogenates.

[0329] For immunohistochemical analysis, after dewaxing and rehydration of the sections, 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). The 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). The 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 of ​​each ROI was calculated.

[0330] To evaluate 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 microhemorrhages will bind to 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.

[0331] 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 ( Fig. 9 In addition, immunohistochemistry using antibody 4G8 showed a 23% (p<0.0001) and 37% (p<0.001) reduction in the hippocampus and cortex, respectively, compared with the isotype control antibody.

[0332] In conclusion, the efficacy of amyloid burden reduction after chronic intraperitoneal injection of BAMB31 in a mouse model of plaque deposition was demonstrated without causing an increased incidence of microhemorrhages, suggesting a favorable ratio of efficacy to toxicity after treatment with the BAMB31 antibody.

[0333] Example 10: Pharmacokinetics of BAMB31 HFA mAb

[0334] The pharmacokinetics of BAMB31 HFA mAb as wild-type IgG1 (BAMB674) versus +YTE IgG1 (BAMB675) isoforms were evaluated in cynomolgus monkeys to directly compare the properties in the peripheral circulation and brain.

[0335] Materials and Methods: Three animals per group were administered 25 mg / kg of each mAb as an intravenous (iv) bolus, and serum samples were collected over a 5-week period. Three additional monkeys were administered 25 mg / kg of each mAb as an iv bolus at week 6, and brain tissue was collected from each group on days 7 and 42 after administration. In total, brain samples were collected from three cynomolgus monkeys on days 7 and 42 for each molecule.

[0336] Drug exposure analysis of BAMB674 and BAMB675 from in vivo cynomolgus monkey serum and brain tissue samples was performed using an electrochemiluminescent immunoassay (ECLIA) method that was individually suitable for the purpose, with endpoint determination performed on a Meso Scale Discovery (MSD) Sector Imager S600. A determination 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 with anti-human Fc specific (CH2 domain) mouse mAb. For brain tissue preparation, homogenates were prepared by freezing and crushing quick-frozen tissue and diluting 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 5-parameter logistic (automatic estimation) fitting and 1 / Y2 standard curve weighting in Watson LIMS software.

[0337] Using a dual compartment (central (V C) and organization (V T ) Compartmental pharmacokinetic (PK) model was used to characterize the PK of BAMB674 and BAMB675 in cynomolgus monkeys. Fig.10 A schematic diagram of the model is shown.

[0338] 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. Fig.11 The half-life of the +YTE IgG1 isotype (BAMB675) was extended by about 1.6-fold compared to the wild-type IgG1 isotype (BAMB674). The FcRn affinity of the +YTE isotype (BAMB675) was increased by about 3-fold compared to the wild-type IgG1 mAb (BAMB674), as shown in Table 5.

[0339] Levels in brain lysates across regions and mAbs were similar at day 7, but YTE mAb was only consistently detected in brain regions and animals at day 42. The results are shown in Fig.12 This is consistent with the increased exposure of the +YTE mAb at later time points.

[0340] In describing the present invention and its various embodiments, for the sake of clarity, specific terms are used. However, the present invention is not intended to be limited to the specific terms so selected. Those skilled in the relevant art will recognize that other equivalent components can be used and other methods can 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 combined.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to human 3pE Aβ, comprising: a. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 22; b. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 10; c. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 12; d. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 13 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 14; e. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 14; f. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 16 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 14; g. a heavy chain variable region consisting of a polypeptide sequence of SEQ ID NO: 20 and a light chain variable region consisting of a polypeptide sequence of SEQ ID NO: 14; h. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 17 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 18; i. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 19 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 18; j. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO: 53; or k. a heavy chain variable region consisting of the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region consisting of the polypeptide sequence of SEQ ID NO:

55.

2. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is chimeric.

3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is human or humanized.

4. An isolated monoclonal antibody that specifically binds to human 3pE Aβ, wherein the heavy chain amino acid sequence and light chain amino acid sequence of the isolated monoclonal antibody are respectively as follows: a. a heavy chain amino acid sequence consisting of SEQ ID NO: 37 and a light chain amino acid sequence consisting of SEQ ID NO: 38; b. a heavy chain amino acid sequence consisting of SEQ ID NO: 39 and a light chain amino acid sequence consisting of SEQ ID NO: 38; c. a heavy chain amino acid sequence consisting of SEQ ID NO: 37 and a light chain amino acid sequence consisting of SEQ ID NO: 52; d. a heavy chain amino acid sequence consisting of SEQ ID NO: 39 and a light chain amino acid sequence consisting of SEQ ID NO: 54; or e. A heavy chain amino acid sequence consisting of SEQ ID NO: 23 and a light chain amino acid sequence consisting of SEQ ID NO:

24.

5. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the monoclonal antibody according to claim 4.

6. A vector comprising the isolated nucleic acid according to claim 5. A host cell comprising the vector according to claim 6 .

8. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the monoclonal antibody according to claim 4 and a pharmaceutically acceptable carrier.

9. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the monoclonal antibody according to claim 4, 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.

10. A method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the monoclonal antibody according to claim 4, the method comprising combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

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