Antibody that binds to AβpE3

Abstract

Disclosed are antibodies or antigen-binding fragments thereof that bind to AβpE3, an N-terminally truncated, pyroglutamic acid-modified form of amyloid beta (Aβ), and their therapeutic and diagnostic uses.

Description

[Technical Field]

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that bind to AβpE3, i.e., N-terminally truncated pyroglutamic acid-modified amyloid beta (Aβ), and their therapeutic and diagnostic uses. [Background technology]

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative dementia disorder that exists in more common late-onset and early-onset familial forms. AD is characterized by the progressive loss of memory and cognitive function. Currently, AD treatment is limited to symptom management, and the prognosis for AD patients is poor. It is estimated that approximately 18 million people worldwide currently suffer from AD, and the number of people suffering from AD is expected to increase due to the aging population. The prevalence of AD doubles approximately every 5 years from the age of 60, from 10% in individuals aged 65 to 50% in individuals aged 85 or older (Non-Patent Document 1).

[0003] Accumulation of Aβ peptides in the brain is thought to play an important role in the neuropathology of AD. Aβ is generated from the amyloid precursor protein (APP) by sequential proteolysis and secreted via major regulatory pathways and, to a lesser extent, constitutive secretory pathways. Aβ is a normal product of cellular metabolism and is present in the plasma and cerebrospinal fluid of healthy individuals. However, abnormal and excessive accumulation of Aβ in the brain leads to the formation of toxic Aβ aggregates that induce synaptic dysfunction and neuronal loss.

[0004] The main variants of Aβ detected in the human brain are Aβ1-40 and Aβ1-42. However, a significant proportion of AD brain Aβ also consists of N-terminally truncated species (Aβn-40 / 42, where n = 2–11). Most of these N-truncated Aβ peptides are thought to be degradation products of full-length Aβ. It has been demonstrated that amyloid aggregates in AD brains and those in cognitively normal elderly subjects have different compositions, and the toxic effects of these aggregates correlate with the predominance of N-terminally truncated species over full-length Aβ. Pyroglutamate-modified Aβ peptides have been demonstrated to be the major component of all N-terminally truncated Aβ species in AD brains. In particular, AβpE3, an Aβ peptide with an amino-terminal pyroglutamate at position 3, has been shown to be the major N-truncated / modified component of intracellular, extracellular, and intravascular Aβ deposits in AD brain tissue. Furthermore, it has been demonstrated that AβpE3 gradually accumulates in the brain in the early stages of AD, even before clinical symptoms appear, suggesting that this peptide plays an important role in the formation of pathological amyloid aggregates. Therefore, N-terminal truncated / modified Aβ peptides are highly desirable and abundant therapeutic targets. This is especially true for AβpE3. For a review and further references, see Non-Patent Document 2.

[0005] Therapeutic antibodies against AβpE3 have been proposed, for example, in US Pat. No. 5,623,299, ... and US Pat. No. 5,623,299.

[0006] Despite the existence of candidate antibodies in the field, no regulatory approved products have yet been developed, and there remains a need in the art for novel therapeutic, preventative, diagnostic and prognostic tools for detecting and treating AD and other neurodegenerative diseases. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2011 / 001366 Pamphlet [Patent Document 2] International Publication No. 2012 / 021469 Brochure [Patent Document 3] International Publication No. 2017 / 123517 Brochure [Patent Document 4] International Publication No. 2018 / 194951 Brochure [Patent Document 5] International Publication No. 2010 / 009987 Pamphlet [Patent Document 6] International Publication No. 2017 / 009459 Brochure [Patent Document 7] International Publication No. 2019 / 149689 Brochure [Patent Document 8] International Publication No. 2020 / 070225 Brochure [Patent Document 9] International Publication No. 2018 / 083628 Brochure [Patent Document 10] International Publication No. 2020 / 193644 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Solomon (2007), Expert Opin Investig Drugs 16(6):819-828 [Non-patent document 2] Perez-Garmendia and Gevorkian (2013), Curr Neuropharmacol 11:491-498 Summary of the Invention [Problem to be solved by the invention]

[0009] One object of the present invention is to provide antibodies or antigen-binding fragments thereof that have novel and useful binding specificities.

[0010] Another object of the present invention is to provide novel candidate antibodies for the treatment of neurodegenerative diseases through targeting AβpE3 peptide, which have beneficial and unique binding profiles.

[0011] Another object of the present invention is to enable the diagnosis of AD and other neurodegenerative disorders through the detection of AβpE3, which is involved in the formation and / or progression of the disease.

[0012] Another object of the present invention is to provide antibodies that bind to AβpE3 peptide with high affinity.

[0013] Another object of the present invention is to provide antibodies that bind to AβpE3 peptide with high specificity.

[0014] Another object of the present invention is to provide antibodies that bind with high selectivity to AβpE3 peptide over other Aβ peptide variants.

[0015] Another object of the present invention is to provide antibodies that bind to the monomeric form of AβpE3 and the putative neurotoxic protofibrillar form that comprises AβpE3.

[0016] Another object of the present invention is to provide antibodies that bind to both the monomeric form of AβpE3 and the putative neurotoxic protofibrils that contain AβpE3.

[0017] Another object of the present invention is to provide antibodies that bind to all forms of AβpE3, including fibrils and plaques that contain AβpE3.

[0018] Another object of the present invention is to provide AβpE3 binding antibodies that combine desirable properties for development into biopharmaceuticals.

[0019] Another object of the present invention is to provide AβpE3 binding antibodies that exhibit little or no immunogenicity upon administration to human subjects.

[0020] Another object of the present invention is to provide AβpE3 binding antibodies that exhibit a favorable pharmacokinetic profile upon administration to human subjects, as evidenced, for example, by one or more of a long half-life, high total exposure, and low clearance. [Means for solving the problem]

[0021] One or more of these objectives, as well as others that will be apparent to one of ordinary skill in the art from reading this entire disclosure, are met by the various aspects disclosed.

[0022] Thus, in a first aspect, the present disclosure provides an antibody or antigen-binding fragment thereof having affinity for AβpE3, wherein the six complementarity-determining regions (CDRs) of the heavy chain variable domain (VH) and the light chain variable domain (VL) consist of the following amino acid sequences: VH-CDR1: GX1TX2N (SEQ ID NO: 1) (In the formula, X1 is selected from Y and F; and X2 is selected from L and M; VH-CDR2: LINPYNGX3TTYNX4KFX5G (SEQ ID NO: 2) (In the formula, X3 is selected from I and V X4 is selected from P and Q; and X5 is selected from M and K; VH-CDR3: EGNWEGVY (SEQ ID NO: 3) VL-CDR1: X6SSQSLLDSNGKTYLH (SEQ ID NO: 4) (In the formula, X6 is selected from K and R; VL-CDR2: LVSX7LDS (SEQ ID NO: 5) (In the formula, X7 is selected from I and K; VL-CDR3: VQGTHFPFT (SEQ ID NO: 6).

[0023] In a second aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention and a pharmaceutically acceptable excipient or carrier.

[0024] In a further aspect, the present invention provides antibodies, antigen-binding fragments thereof and / or pharmaceutical compositions comprising same for use in the methods of treatment or in the methods of detection or diagnosis described herein.

[0025] Anti-AβpE3 antibody As described above, in a first aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that has affinity for AβpE3 and in which the six CDRs of the VH and VL domains are as defined above with respect to SEQ ID NOs: 1 to 6.

[0026] The present invention is based on detailed insights into the pathophysiology of diseases characterized by amyloid aggregation and the identification of specific forms of Aβ in brain tissue from patients suffering from such diseases. As a non-limiting example, soluble forms of AβpE3 have been found in extracts from AD brains, further highlighting the importance of obtaining antibodies that bind to such species in a specific and / or selective manner. However, these findings also point to the potential benefits of having antibodies that bind to all forms of AβpE3 present in disease associations. These findings have enabled the generation of the present antibodies or antigen-binding fragments thereof that are specific and / or selective for AβpE3 in its various forms. It has also enabled the further development of the initial antibodies into humanized antibodies and variants thereof that possess several beneficial properties, including unexpectedly favorable pharmacokinetic profiles. The generation and characterization of exemplary such antibodies are detailed in Examples 1-14.

[0027] Without being bound by theory, it is believed that such novel antibodies or antigen-binding fragments thereof are useful for the diagnosis, prognosis, and / or treatment of neurodegenerative diseases such as AD through specific binding to the putative disease-causing Aβ variant AβpE3.

[0028] As defined herein, embodiments of antibodies or antigen-binding fragments thereof of the first aspect of the disclosure are characterized by specific amino acid sequences in the regions that determine their binding ability, such as the CDRs of the heavy and / or light chain variable domains, or indeed the entire VL and / or VH domains or regions. Non-limiting examples of such specific amino acid sequences are provided herein for the specific antibodies generated as described in Examples 1-14. It is believed that the specific sequence information provided for the generated antibodies will enable one of skill in the art to define combinations and variations of these sequences that are within the scope of the invention.

[0029] Thus, in one embodiment of the first aspect, the antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2 and VL-CDR2 regions consisting of the following amino acid sequences: VH-CDR1: GFTMN (SEQ ID NO: 7) VH-CDR2: LINPYNGVTTYNQKFKG (SEQ ID NO: 8) VL-CDR2: LVSILDS (SEQ ID NO: 9).

[0030] In more specific embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 22 and an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 22, provided that the three VH-CDR regions consist of SEQ ID NOs: 7, 8, and 3. For example, the amino acid sequence contained in the VH of such an antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 15 to 21, such as selected from the group consisting of SEQ ID NOs: 15 to 16 and 18 to 21, or selected from the group consisting of SEQ ID NOs: 15 to 16 and 18 to 20, particularly selected from the group consisting of SEQ ID NOs: 15 and 18, most particularly SEQ ID NO: 18, or selected from the group consisting of SEQ ID NOs: 15 to 20, such as a sequence having at least 80% identity to any one of the enumerated subgroups, provided always that the three VH-CDR regions consist of SEQ ID NOs: 7, 8, and 3.

[0031] In another embodiment, the antibody or antigen-binding fragment thereof has the amino acid sequence RSSQSLLDSNGKTYLH (SEQ ID NO: 10) and a VL-CDR1 consisting of:

[0032] In more specific such embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 24 and an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 23 to 24, provided that the three VL-CDR regions consist of SEQ ID NOs: 10, 9, and 6. For example, the amino acid sequence contained in the VL of such an antibody or antigen-binding fragment thereof is SEQ ID NO: 23 or a sequence having at least 80% identity to SEQ ID NO: 23.

[0033] In one embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 22 and an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 22 (provided that the three VH-CDR regions consist of SEQ ID NOs: 7, 8, and 3); and It comprises both a light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 24 and an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 23 to 24 (provided that the three VL-CDR regions consist of SEQ ID NOs: 10, 9 and 6).

[0034] For example, the antibody or antigen-binding fragment thereof of the first aspect may comprise a VH / VL combination selected from the group consisting of: a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; f) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; g) a heavy chain variable domain comprising SEQ ID NO: 21 and a light chain variable domain comprising SEQ ID NO: 24; and h) a heavy chain variable domain comprising SEQ ID NO: 22 and a light chain variable domain comprising SEQ ID NO: 23;

[0035] for example, a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; f) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; and g) a heavy chain variable domain comprising SEQ ID NO: 21 and a light chain variable domain comprising SEQ ID NO: 24 From the group consisting of VH / VL combinations of

[0036] or a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; and g) a heavy chain variable domain comprising SEQ ID NO: 21 and a light chain variable domain comprising SEQ ID NO: 24 From the group consisting of VH / VL combinations of

[0037] for example, a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; and f) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23 From the group consisting of VH / VL combinations of

[0038] for example, a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; and e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23 From the group consisting of VH / VL combinations of

[0039] for example, a) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23 and a heavy chain variable domain and a light chain variable domain selected from the group consisting of the VH / VL combinations

[0040] For example, the antibody or antigen-binding fragment thereof may be a) comprising a VH / VL combination of a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23 The invention comprises a heavy chain variable domain and a light chain variable domain selected from:

[0041] In another embodiment of the first aspect, the present disclosure provides a method for the production of a VL-CDR1 polypeptide comprising the steps of: An antibody or antigen-binding fragment thereof is provided, which is KSSQSLLDSNGKTYLH (SEQ ID NO: 11).

[0042] In a more specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence SEQ ID NO:25 or an amino acid sequence having at least 80% identity to SEQ ID NO:25, with the proviso that the three VH-CDR regions consist of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:3.

[0043] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain comprises the amino acid sequence SEQ ID NO:26 or an amino acid sequence having at least 80% identity to SEQ ID NO:26, provided that the three VL-CDR regions consist of SEQ ID NO:11, SEQ ID NO:9 and SEQ ID NO:6.

[0044] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:25 or an amino acid sequence having at least 80% identity to SEQ ID NO:25, provided that the three VH-CDR regions consist of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:3; and It comprises both a light chain variable domain comprising the amino acid sequence SEQ ID NO:26 or an amino acid sequence having at least 80% identity to SEQ ID NO:26 (provided that the three VL-CDR regions consist of SEQ ID NO:11, SEQ ID NO:9 and SEQ ID NO:6).

[0045] In another embodiment of the first aspect, the disclosure provides an antibody or antigen-binding fragment thereof, wherein the VH-CDR1, VH-CDR2, VL-CDR1 and VL-CDR2 regions have the following amino acid sequences: VH-CDR1: GYTLN (SEQ ID NO: 12); VH-CDR2: LINPYNGITTYNPKFMG (SEQ ID NO: 13); VL-CDR1: KSSQSLLDSNGKTYLH (SEQ ID NO: 11) VL-CDR2: LVSKLDS (SEQ ID NO: 14) The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0046] In a more specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the amino acid sequence SEQ ID NO:27 or an amino acid sequence having at least 80% identity to SEQ ID NO:27, with the proviso that the three VH-CDR regions consist of SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:3.

[0047] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain comprises the amino acid sequence SEQ ID NO:28 or an amino acid sequence having at least 80% identity to SEQ ID NO:28, provided that the three VL-CDR regions consist of SEQ ID NO:11, SEQ ID NO:14 and SEQ ID NO:6.

[0048] In yet another embodiment, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:27 or an amino acid sequence having at least 80% identity to SEQ ID NO:27, with the proviso that the three VH-CDR regions consist of SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:3; and It comprises both a light chain variable domain comprising the amino acid sequence SEQ ID NO:28 or an amino acid sequence having at least 80% identity to SEQ ID NO:28 (provided that the three VL-CDR regions consist of SEQ ID NO:11, SEQ ID NO:14 and SEQ ID NO:6).

[0049] In certain embodiments, the definition of the VH and VL sequences of the antibody or antigen-binding fragment thereof is limited to any one of the listed sequences and sequences having at least 85%, such as at least 90%, for example at least 95%, for example at least 98%, such as at least 100% identity thereto.

[0050] In a specific embodiment, the VH / VL combinations are those present in the antibodies exemplified in Examples 1-13 (see especially Tables 1, 6 and 12).

[0051] In embodiments in which the variable domain of an antibody or antigen-binding fragment is defined by a particular percentage of sequence identity to a reference sequence, the VH and / or VL domain retains identical CDR sequences as present in the reference sequence, such that variations are present only in the framework regions.

[0052] In one embodiment of the antibody or antigen-binding fragment thereof according to the first aspect, the CDR regions are as defined using the Kabat convention, which is well known to those skilled in the art of antibody technology (see, e.g., Kabat (1991), Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 5 th (See NIH Publication no. 91-3242, 2002 edition).

[0053] As those skilled in the art will recognize, Aβ peptides exist in various lengths and forms. Of particular relevance to the present disclosure is the N-terminus of the Aβ peptide, in that the antibodies herein bind to Aβ peptides that have a truncation of two amino acid residues compared to full-length Aβ (i.e., Aβ peptides beginning with an aspartic acid residue, commonly designated "D1"). On the other hand, the C-terminus of the peptide is of little relevance to the present disclosure; as is well known, Aβ peptides may be 43 amino acid residues long or may already terminate at, for example, position 28. As used herein, the term "AβpE3" encompasses any Aβ peptide beginning with a pyroglutamic acid ("pE3") residue corresponding to the third amino acid residue in the full-length peptide, regardless of its C-terminus, whether or not it has been truncated with respect to the full-length sequence. Similarly, "Aβ1-X" is used interchangeably with "full-length Aβ" and refers to any Aβ peptide beginning with the first amino acid residue (i.e., D1), regardless of its C-terminus.

[0054] Those skilled in the art also recognize that Aβ peptides can exist in various forms during their progressive aggregation from monomers to insoluble plaques. Of particular relevance to the present disclosure, soluble forms of Aβ peptides can exist in monomeric form or in various oligomeric or further aggregated forms. Soluble forms of polymerized or aggregated Aβ peptides are collectively referred to as "protofibrils" in this disclosure. For clarity, "protofibrils" is intended to encompass oligomers and higher-order aggregates, but exclude insoluble fibrils or amyloid plaques.

[0055] In certain embodiments, the antibody or antigen-binding fragment thereof according to the first aspect has affinity for AβpE3 in a form selected from the group consisting of monomers, protofibrils, fibrils, and plaques. In more specific embodiments, the antibody or antigen-binding fragment thereof has affinity for AβpE3 in a form selected from monomers and protofibrils. In yet another embodiment, the antibody or antigen-binding fragment thereof has affinity for AβpE3 monomers. In a further embodiment, the antibody or antigen-binding fragment thereof has affinity for AβpE3 protofibrils. It is noted that the antibody or antigen-binding fragment thereof according to the first aspect may have affinity for AβpE3 in general, for example, AβpE3 monomers as well as AβpE3 protofibrils and / or other AβpE3 species.

[0056] Alternatively, the antibody or antigen-binding fragment thereof may exhibit a preference or selectivity for one form of AβpE3 over another form of AβpE3. In one such embodiment, the antibody or antigen-binding fragment thereof has a higher binding affinity for AβpE3-containing protofibrils than for AβpE3 monomers. Without wishing to be bound by theory, such higher affinity for protofibrils in embodiments of the antibody or fragment thereof may be due to an avidity effect, inasmuch as the protofibrillar form of Aβ exhibits multiple epitopes to which the antibody binds compared to the monomeric form. Thus, the affinity of an antibody for protofibrils may be measured by methods known to those skilled in the art and reported herein as "apparent affinity." In one embodiment, the antibody or antigen-binding fragment thereof has a binding affinity that is at least 2-fold higher, e.g., at least 10-fold higher, e.g., at least 40-fold higher, e.g., at least 100-fold higher, e.g., at least 200-fold higher, for AβpE3-containing protofibrils than for AβpE3 monomers.

[0057] In certain embodiments, the antibody and antigen-binding fragment thereof of the first aspect selectively binds to AβpE3. As used herein, the term "selectively binds" refers to preferential binding of the antibody or antigen-binding fragment thereof to the AβpE3 target. In certain embodiments, the antibody and antigen-binding fragment thereof of the first aspect does not bind to any appreciable extent to uncleaved amyloid beta Aβ1-X. Stated slightly differently, in one such embodiment, the antibody or antigen-binding fragment thereof has a higher binding affinity for AβpE3 monomer than for Aβ1-X monomer. In more specific embodiments, the antibody or antigen-binding fragment thereof has a binding affinity that is at least 2-fold higher, e.g., at least 10-fold higher, e.g., at least 100-fold higher, e.g., at least 1000-fold higher, e.g., at least 3000-fold higher, for AβpE3 than for Aβ1-X monomer.

[0058] As used herein, the terms "specific binding to X," "selective binding to X," and "affinity for X," where X is an antigen or epitope, refer to properties of an antibody or antigen-binding fragment thereof that can be tested by, for example, ELISA, surface plasmon resonance (SPR), equilibrium exclusion binding (KinExA®), or biolayer interferometry (BLI). Those of skill in the art will be aware of these and other methods.

[0059] For example, binding affinity for antigen or epitope X can be tested in an experiment in which the antibody or antigen-binding fragment thereof to be tested is captured on an ELISA plate coated with antigen X or an antigen representing epitope X, a biotinylated detection antibody is added, followed by streptavidin-conjugated horseradish peroxidase (HRP). Alternatively, the detection antibody may be directly conjugated to HRP. Tetramethylbenzidine (TMB) substrate is added, and absorbance at 450 nm is measured using an ELISA multiwell plate reader. One skilled in the art can then interpret the results obtained from such an experiment to establish at least a qualitative measure of the binding affinity of the antibody or antigen-binding fragment thereof to X. ELISA can also be used when a quantitative measure is desired, for example, to determine the EC50 value (half maximal effective concentration) for the interaction. The response of the antibody or antigen-binding fragment thereof to a dilution series of X can be measured using ELISA as described above. One skilled in the art can then interpret the results obtained from such experiments and an EC50 value can be calculated from the results using, for example, GraphPad Prism v.9 and non-linear regression.

[0060] As used herein, the term "EC50" refers to the half-maximal effective concentration of an antibody or antigen-binding fragment thereof that induces a response halfway between baseline and maximum after a specified exposure time.

[0061] Furthermore, inhibition ELISAs can be used to obtain a quantitative measure of interaction by determining the "IC50" (half maximal inhibitory concentration). In inhibition ELISAs, the concentration of antigen or epitope X in a fluid sample is measured by detecting interference with the expected signal output. In principle, a known antigen- or epitope-bearing substance is used to coat a multiwell plate. In parallel, an antibody or its antigen-binding fragment, suspected to have affinity for the antigen or epitope, is added and incubated with solutions containing various concentrations of the antigen. Following standard blocking and washing steps, a sample containing a mixture of the antibody or its antigen-binding fragment and the antigen or epitope is added to the well. Next, a labeled detection antibody with affinity for the antigen- or epitope-binding antibody or its antigen-binding fragment is applied for detection using a relevant substrate (e.g., TMB). In principle, if a high concentration of antigen or epitope is present in the fluid sample, a significant decrease in signal output will be observed. In contrast, if the antigen or epitope is very low in the fluid sample, there will be little decrease in the expected signal output. Those skilled in the art will appreciate that the signal output also depends on the affinity of the antibody or antigen-binding fragment thereof for said antigen or epitope.

[0062] As used herein, the term "IC50" refers to the half-maximal inhibitory concentration of an antibody or antigen-binding fragment thereof that induces a response intermediate between the baseline and the maximum inhibition after a specified exposure time. As used herein, a lower IC50 value indicates that a lower concentration of antigen or epitope is required to block the binding of the detection antibody to the known antigen or epitope coated on the plate, compared to a higher IC50 value. Thus, a lower IC50 value typically corresponds to a higher affinity.

[0063] The binding affinity of an antibody or antigen-binding fragment thereof can also be tested by SPR. For example, the binding affinity can be tested in an experiment in which an antigen or epitope X is immobilized on a sensor chip of an instrument, and a sample containing the antibody or antigen-binding fragment thereof to be tested is passed over the chip. Alternatively, the antibody or antigen-binding fragment thereof to be tested can be immobilized on a sensor chip of an instrument, and a sample containing X is passed over the chip. One skilled in the art can then interpret the results obtained by such an experiment to establish at least a qualitative measure of the binding affinity of the moiety for X. For example, the K of the interaction D SPR can also be used if a quantitative measure is desired to determine the binding value. Binding values ​​can be determined, for example, with a Biacore (Cytiva) or ProteOn XPR 36 (Bio-Rad) instrument. The antigen or epitope is suitably immobilized on the instrument's sensor chip, and a sample of the antibody or antigen-binding fragment thereof whose affinity is to be determined is prepared by serial dilution and injected. Then, K D Values ​​can be calculated from the results using, for example, the 1:1 Langmuir binding model in Biacore Insight Evaluation Software 2.0 or other suitable software, typically provided by the instrument manufacturer.

[0064] Another method for determining the binding affinity of an antibody or antigen-binding fragment thereof to an antigen or epitope X is the equilibrium exclusion method (KinExA; Sapidyne Instruments Inc; Darling and Brault, Assay and Drug Dev Tech (2004) 2(6):647-657) for measuring equilibrium binding affinities and kinetics between unmodified molecules in solution. D The assay requires immobilization of one interaction partner (e.g., the adjusted binding partner) on a solid phase, which is then used as a probe to capture the other interaction partner (e.g., the constant binding partner), which is free and in solution once equilibrium is reached.

[0065] Binding affinity can also be measured by biolayer interferometry (BLI), a label-free technique for measuring biomolecular interactions within the interactome. This is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized proteins on a biosensor chip and an internal reference layer. Binding between a ligand (antigen or epitope X) immobilized on the biosensor chip surface and an analyte in solution (such as an antibody or antigen-binding fragment thereof with affinity for X) results in an increase in optical thickness at the biosensor chip, which results in a wavelength shift Δλ, which is a direct measure of the change in thickness of the biological layer. The interaction is measured in real time, providing the ability to precisely and accurately monitor binding specificity, association and dissociation rates, or concentration.

[0066] Those skilled in the art will be aware of these and other methods for determining the affinity of an antibody or antigen-binding fragment thereof for an antigen or epitope X, either qualitatively or quantitatively, or both.

[0067] In one embodiment of the antibody or antigen-binding fragment thereof, it has a K of 1 nM or less, e.g., 1-200 pM, e.g., 10-100 pM, as determined by SPR. D The binding affinity (or apparent binding affinity) for AβpE3-containing protofibrils corresponds to a value of 0.05.

[0068] In another embodiment, the antibody or antigen-binding fragment thereof has a K of 100 nM or less, for example, 0.1 to 50 nM, for example, 0.5 to 10 nM, as determined by SPR. D The binding affinity for AβpE3 monomers corresponds to the value.

[0069] In some embodiments of the antibody or antigen-binding fragment thereof according to the first aspect, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a single-chain Fv fragment, an (scFv)2, and a domain antibody. In one embodiment, the at least one antibody or antigen-binding fragment thereof is selected from a full-length antibody, a Fab fragment, and an scFv fragment. In one particular embodiment, the antibody is a full-length antibody.

[0070] In one embodiment, the antibody or antigen-binding fragment thereof is of the IgG class. In a more specific embodiment, the antibody or antigen-binding fragment thereof is of a subclass selected from IgG1 and IgG4.

[0071] In one embodiment, the antibody or antigen-binding fragment thereof is monoclonal.

[0072] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, an antibody that has been mutated to reduce its antigenicity in humans, and an antigen-binding fragment thereof.

[0073] As used herein, the term "antibody or antigen-binding fragment thereof" encompasses not only full-length or intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof, such as Fab, Fab', F(ab')2, Fab3, Fv and variants thereof, fusion proteins comprising one or more antibody portions, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of the antibody, amino acid sequence variants of the antibody, and covalently modified antibodies. Further examples of modified antibodies and antigen-binding fragments thereof include nanobodies, AlbudAbs, DARTS (dual affinity retargeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFS (dual-acting Fabs), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-lgs (dual variable domain immunoglobulins), CovX-bodies (peptide-modified antibodies), duobodies, and triomAbs. This list of variants of antibodies and antigen-binding fragments thereof should not be considered limiting, and those skilled in the art will recognize other suitable variants.

[0074] A full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). Depending on the amino acid sequence of the constant domain of the antibody's heavy chain, antibodies are assigned to different classes. There are six major classes of antibodies: IgA, IgD, IgE, IgG, IgM, and IgY, some of which can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgG1, and IgG2. As used herein, the term "full-length antibody" refers to an antibody of any class, such as IgD, IgE, IgG, IgA, IgM, or IgY (or any subclass thereof). The subunit structures and three-dimensional configurations of different classes of antibodies are well known.

[0075] The term "antigen-binding fragment" refers to a portion or region of an antibody molecule or derivative thereof that retains all or a substantial portion of the antigen-binding ability of the corresponding full-length antibody. An antigen-binding fragment may include a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL regions or domains typically contains three CDRs, i.e., CDR1, CDR2, and CDR3, with CDRs from the VH domain designated as VH-CDR1, VH-CDR2, and VH-CDR3, and CDRs from the VL domain designated as VL-CDR1, VL-CDR2, and VL-CDR3. The three CDRs in VH or VL are flanked by framework regions (FR1, FR2, FR3, and FR4). As briefly listed above, examples of antigen-binding fragments include, but are not limited to, (1) Fab fragments, which are monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) Fab' fragments, which are Fab fragments having a heavy chain hinge region; (3) F(ab')2 fragments, which are dimers of Fab' fragments linked by heavy chain hinge regions, for example, linked by disulfide bridges at the hinge regions; (4) Fc fragments; (5) Fv fragments, which are the minimum antibody fragments having the VL and VH domains of a single arm of an antibody; (6) single-chain Fv (scFv) fragments, which are single polypeptide chains in which the VH and VL domains of scFv are linked by a peptide linker; (7) (scFv)2, which comprises two VH domains and two VL domains linked via disulfide bridges via the two VH domains; and (8) domain antibodies, which can be antibody single variable domain (VH or VL) polypeptides that specifically bind to an antigen. Antigen-binding fragments can be prepared by conventional methods. For example, F(ab')2 fragments can be produced by pepsin digestion of full-length antibody molecules, and Fab fragments can be produced by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, portions can be prepared recombinantly by expressing heavy and light chain portions in suitable host cells (e.g., E. coli, yeast, mammalian, plant, or insect cells) and allowing them to assemble to form the desired antigen-binding fragment, either in vivo or in vitro.Single-chain antibodies can be prepared recombinantly by linking a nucleotide sequence encoding a heavy chain variable region with a nucleotide sequence encoding a light chain variable region, for example, by incorporating a flexible linker between the two variable regions.

[0076] Furthermore, those skilled in the art are aware of the meaning of the terms polyclonal antibody and monoclonal antibody. Polyclonal antibodies are typically produced by administering an antigen to an animal. The antigen will induce an immune response that results in polyclonal antibodies. Monoclonal antibodies are produced by immunizing an animal, usually a mouse, with an antigen and then isolating the spleen from the animal. The isolated spleen cells are immortalized by fusion with myeloma cells to produce hybridoma cells. Each hybridoma cell produces a unique monoclonal antibody.

[0077] As used herein, the term "human antibody" refers to an antibody having variable and constant regions corresponding to or derived from an antibody obtained from a human subject. As used herein, the term "chimeric antibody" refers to a recombinant or genetically engineered antibody, such as an antibody having variable regions (VH and VL) of murine origin and a human constant region (Fc), to reduce the immunogenicity of the antibody. The term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans, in order to reduce the immunogenicity of the intact antibody itself.

[0078] Pharmaceutical Composition In a second aspect, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and at least one pharmaceutically acceptable excipient or carrier.

[0079] Techniques for formulating antibodies for human therapeutic use are well known in the art and are described, for example, in Wang et al. (2007), J Pharm Sci, 96:1-26, the contents of which are incorporated herein in their entirety.

[0080] Pharmaceutically acceptable excipients that can be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0081] In certain embodiments, the pharmaceutical composition is formulated for administration to a subject by any suitable route of administration, including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal administration. In preferred embodiments, the composition is formulated for intravenous or subcutaneous administration.

[0082] Methods of prevention, treatment, diagnosis, prognosis and detection Antibodies or antigen-binding fragments thereof according to the present disclosure may be useful as therapeutic and / or diagnostic agents.

[0083] Thus, in a further aspect of the disclosure there is provided an antibody or antigen-binding fragment thereof according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a medicament.

[0084] In yet another aspect of the present disclosure, there is provided an antibody or antigen-binding fragment thereof according to the first aspect, or a pharmaceutical composition according to the second aspect, for use as a diagnostic agent.

[0085] Also provided are methods for preventing, treating, or diagnosing a disease, or assessing the prognosis of a disease, in which an antibody or antigen-binding fragment thereof disclosed herein is administered to a subject, typically a human subject.

[0086] Also provided is the use of the disclosed antibodies or antigen-binding fragments thereof for the manufacture of a composition (such as a medicament) for use in the prevention, treatment, diagnosis and / or prognosis of any one of the listed diseases.

[0087] Also provided are methods for detecting or diagnosing disease in a subject, comprising contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof described herein. These methods are typically in vitro methods.

[0088] Therefore, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, is useful for treating, preventing, and / or diagnosing a condition selected from neurological disorders or conditions characterized by the accumulation and / or aggregation of Aβ, such as amyloid plaque formation, including Alzheimer's disease (AD) (including familial and sporadic AD), mild cognitive impairment (MCI), dementia with Lewy bodies, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), and other diseases based on or associated with amyloidogenic proteins, such as progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jacob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataracts due to Aβ deposition, traumatic brain injury associated with Aβ accumulation, adult-onset diabetes, senile cardiac amyloidosis, and macular degeneration.

[0089] Thus, in one embodiment, there is provided an antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, for use in the treatment, prevention, diagnosis, and / or prognosis of an Aβ peptide-related pathology selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), dementia with Lewy bodies, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataracts caused by Aβ deposition, traumatic brain injury accompanied by Aβ accumulation, adult-onset diabetes, senile cardiac amyloidosis, and macular degeneration.

[0090] In one particular embodiment, the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising same, is provided for use in the treatment, prevention, diagnosis and / or prognosis of Alzheimer's disease.

[0091] In another aspect, there is provided a method for the treatment, prevention, diagnosis and / or prognosis of an Aβ peptide-related pathology in a mammal having or at risk of developing said disorder, comprising administering to said mammal an amount, such as a therapeutically effective amount, of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising same.

[0092] In one embodiment, the Aβ peptide-related pathology is selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), dementia with Lewy bodies, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataracts caused by Aβ deposition, traumatic brain injury accompanied by Aβ accumulation, adult-onset diabetes, senile cardiac amyloidosis, and macular degeneration. In a more specific embodiment, the Aβ peptide-related pathology is Alzheimer's disease.

[0093] There are several putative mechanisms of action for the therapeutic or prophylactic use of the disclosed antibodies or antigen-binding fragments thereof for the treatment of neurodegenerative diseases. Without being bound by theory, non-limiting and independently possible mechanisms of action include, for example, preventing the dissemination of Aβ aggregation by binding to AβpE3 monomers, binding to and removing soluble neurotoxic AβpE3-containing aggregates (protofibrils), and / or clearing AβpE3-containing amyloid plaques, thereby attenuating amyloidosis and improving cognitive function.

[0094] Regarding the diagnostic or prognostic use of the disclosed antibodies or antigen-binding fragments thereof in neurodegenerative diseases, putatively harmful AβpE3 species can be detected and measured in patients at risk for or showing early signs of disease. One such method is a PET scan using a radiolabeled antibody of the present disclosure. Another method for diagnosis and prognosis is biochemical analysis, analyzing levels of AβpE3 in blood, plasma, CSF, and other body fluids using methods such as ELISA, Mesoscale Discovery (MSD), or Simoa.

[0095] Incorporation by Reference Various publications are cited in this application, each of which is incorporated herein by reference in its entirety. [Brief explanation of the drawings]

[0096] [Figure 1] Binding of the indicated hybridoma clones to monomeric Aβ1-42, Aβ2-42, Aβ3-42, AβpE3-42, and Aβ4-42 using ELISA as described in Example 1 is shown. [Figure 2] Binding of the indicated hybridoma clones to AβpE3-42 protofibrils using ELISA as described in Example 1 is shown. [Figure 3] 1 shows inhibition-response curves of the indicated recombinant antibodies against monomeric AβpE3-40 using an inhibition ELISA as described in Example 3. [Figure 4] 1 shows inhibition-response curves of the indicated recombinant antibodies against monomeric Aβ1-40 using an inhibition ELISA as described in Example 3. [Figure 5] 1 shows inhibition-response curves of the indicated recombinant antibodies against AβpE11-40 using inhibition ELISA as described in Example 3. [Figure 6] 1 shows inhibition-response curves of the indicated recombinant antibodies against AβpE3-42 protofibrils (PF) using inhibition ELISA as described in Example 3. [Figure 7] 1 shows the binding interactions of the indicated recombinant antibodies to AβpE3-40 monomer as measured by SPR as described in Example 3. [Figure 8] 1 shows the binding interactions of the indicated recombinant antibodies to AβpE3-42 protofibrils measured by SPR as described in Example 3. [Figure 9] FIG. 1 shows depletion of AβpE3-40 levels in soluble AD brain extracts by the indicated recombinant antibodies using immunoprecipitation, as described in Example 4. [Figure 10] FIG. 1 shows depletion of AβpE3-42 levels in soluble AD brain extracts by the indicated recombinant antibodies using immunoprecipitation, as described in Example 4. [Figure 11] Binding of the indicated recombinant antibodies to amyloid plaques in human brain sections by immunohistochemistry as described in Example 4. [Figure 12] Binding of the indicated humanized antibodies to monomeric AβpE3-28, Aβ1-28, Aβ2-28, Aβ3-28, Aβ4-28, Aβ5-28, and AβpE11-28 using an inhibition ELISA as described in Example 6 is shown. [Figure 13] Binding of the indicated humanized antibodies to AβpE3-40 and Aβ1-40 monomers and AβpE3-42 and Aβ1-42 protofibrils using an inhibition ELISA as described in Example 6 is shown. [Figure 14] 1 shows the binding interactions of the indicated humanized antibodies to AβpE3-40 monomer as measured by SPR, as described in Example 6. [Figure 15] 1 shows the binding interactions of the indicated humanized antibodies to AβpE3-42 protofibrils measured by SPR as described in Example 6. [Figure 16] FIG. 1 shows immunoprecipitation of AβpE3-x levels in soluble AD brain extracts with the indicated humanized antibodies using immunoprecipitation as described in Example 7. [Figure 17] Binding of the indicated humanized antibodies to amyloid plaques in human brain sections by immunohistochemistry as described in Example 7. [Figure 18] 1 shows the concentration-response effect of the indicated humanized antibodies on the aggregation of AβpE3, as described in Example 8. [Figure 19] 1 shows the effect of the indicated humanized antibodies on Aβ plaque clearance in AD brain sections as described in Example 8. [Figure 20] 1 shows the pharmacokinetic plasma concentration-time profiles in mice of the indicated murine and humanized antibodies as described in Example 9. [Figure 21] 1 shows the pharmacokinetic plasma concentration-time profiles in mice of the indicated humanized antibodies as described in Example 10. [Figure 22] 1 shows the binding interactions of the indicated humanized antibodies to AβpE3-40 monomer as measured by SPR, as described in Example 11. [Figure 23]1 shows the binding interactions of the indicated humanized antibodies to AβpE3-42 protofibrils measured by SPR as described in Example 11. [Figure 24] FIG. 10 shows immunoprecipitation of AβpE3-x levels in soluble AD brain extracts with the indicated humanized antibodies using immunoprecipitation as described in Example 12. [Figure 25] 1 shows binding of the indicated humanized antibodies to amyloid plaques in human brain sections by immunohistochemistry, as described in Example 12. [Figure 26] 1 shows the concentration-response effect of the indicated humanized antibodies on the aggregation of AβpE3, as described in Example 13. [Figure 27] 1 shows the effect of the indicated humanized antibodies on Aβ plaque clearance in AD brain sections, as described in Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0097] Example While the present invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for those elements without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope of the invention. Therefore, it is not intended that the present invention be limited to any particular embodiment, but rather that the invention includes all embodiments falling within the scope of the appended claims. The present invention is further illustrated by the following non-limiting examples. [Example]

[0098] Example 1 Generation and screening of antibodies against AβpE3 This example describes the immunization of BALB / c mice and the subsequent generation and screening of hybridoma cell lines.

[0099] Materials and Methods Immunogen preparation: Protofibrils prepared from the AβpE3-42 peptide were used as the immunogen. Briefly, the AβpE3-42 peptide (American Peptide) was dissolved in 10 mM NaOH, pH >11, at a concentration of 100 μM. Protofibrils were prepared by neutralizing the AβpE3-42 peptide to pH 7.4 by adding 1:1 2x PBS buffer to a final concentration of 50 μM. For protofibril formation, the peptide was incubated at 37°C for 20 min and purified from residual monomers by HPLC on a Superdex 75 10 / 300GL size-exclusion column using a mobile phase of 1x PBS, 0.1% Tween-20, pH 7.4. Prior to injection, the protofibril reaction was centrifuged at 16,000 x g for 5 min at 4°C. The fibrillar material was pelleted, and the supernatant contained soluble AβpE3-42 protofibrils and monomers. Protofibrils were eluted into the excluded volume of the column, and the void fraction was collected. Repeated injections and collection of the void peak were performed to obtain sufficient material for immunization. A total of 14 injections were performed, and 44 ml was collected and concentrated to a final concentration of 20 μm using a 50 MWCO Amicon filter (UFC805024, Millipore). Briefly, the Amicon filter was prewetted in PBS, 0.005% Tween-20, pH 7.4 for 2 hours before sample loading. Protofibrils were added and concentrated 8-fold by centrifugation at 3200 × g. Purified protofibrils were stored at -80°C until use.

[0100] Immunization: Eight-week-old BALB / c mice (n = 3) were immunized with AβpE3-42 protofibrils prepared as described above. AβpE3-42 protofibrils at a concentration of approximately 20 μM (86 μg / ml) were placed in sterile PBS, pH 7.4, containing 0.1% Tween-20. For each immunization, 400 μl of AβpE3-42 protofibrils (approximately 34 μg) were mixed with 5 μl of ISCOM adjuvant (12 μg), and 200 μl was administered subcutaneously on each side. Each mouse received three or four immunizations. Plasma samples were collected 2–3 weeks after each immunization. When antibodies reactive to AβpE3-40 with an endpoint titer of approximately 1 / 100,000 were detected in the plasma, one final booster injection (immunogen without ISCOM adjuvant) was administered intraperitoneally. After 3 days, the mice were sacrificed and the spleens were harvested.

[0101] Tissue collection: Blood was collected from the tail vein at all time points except at sacrifice, when blood was collected from the heart. For sacrifice, mice were anesthetized with 300 mg / kg ketamine and 4 mg / kg medetomidine, and blood was collected from the right atrium into a Microtainer tube. The sample was then centrifuged at 2400 × g for 10 minutes, and the plasma was transferred to a pre-labeled low-binding Eppendorf tube. Plasma samples were immediately frozen on dry ice and stored at -80 °C. Spleens were collected by opening the abdominal cavity and isolating the intact spleen. The spleen was placed in a 15 ml tube containing 5 ml DMEM medium (2 × PenStrep) at room temperature (RT) and transported on ice within 1 hour for preparation of a spleen cell suspension.

[0102] Plasma screening by direct ELISA: Plasma samples were analyzed for reactivity to AβpE3-40 monomer after each immunization by direct ELISA to determine when to stop immunization and initiate hybridoma generation. AβpE3-40 peptide (Anaspec) was dissolved at 100 μM in 10 mM NaOH + 0.005% Tween-20 and stored in aliquots at -80°C. For coating, freshly thawed 100 μM AβpE3-40 peptide was diluted in PBS to prepare a 0.5 μM solution, and ELISA microtiter plates were coated with 50 μl of AβpE3-40 per well overnight at +4°C. The plate was washed four times with 1x ELISA wash buffer (containing 0.28 mM NaH2PO4, 2.5 mM Na2HPO4, 150 mM NaCl, 0.1% Tween-20, and 0.0075% Kathon CG), followed by adding 100 μl / well of Pierce blocking buffer and incubating at room temperature for 1 hour with shaking (900 rpm) to block residual binding capacity. After discarding the blocking buffer, samples (mouse plasma) or standards were added to each well. Plasma samples from immunized mice were diluted at least 1:1000 and further diluted 1:2 in seven steps in the wells. Plasma from unimmunized mice was used as a negative control. A standard curve was generated using the control antibody mAb 6E10 (Covance #SIG-39320, epitope: amino acids 1-16 of Aβ) with two-fold serial dilutions starting at 1 ng / ml (final concentration range: 0.016–1 ng / ml). Samples were incubated at room temperature for 90 minutes with shaking (900 rpm). The plate was washed as above, and 50 μl of HRP-conjugated anti-mouse IgG (diluted 1 / 10,000 in incubation buffer consisting of 1x Dulbecco's PBS containing 0.1% BSA and 0.05% Tween-20) was added to each well, followed by incubation at room temperature for 1 hour with shaking (900 rpm). For detection, the plate was washed again as above, and 50 μl of room-temperature TMB was added to each well. The plate was then incubated at room temperature for 15 minutes without shaking.The reaction was stopped by adding 50 μl of 2 M H2SO4 and the plates were read within 15 minutes at a wavelength of 450 nm. An endpoint titer of 1 / 100,000 was considered sufficiently high, and no further immunizations were performed after this was reached.

[0103] Hybridoma generation: Splenocytes isolated from sacrificed mice were fused with cells from an immortalized cell line (SP2 / 0) to generate hybridomas. Briefly, single-cell suspensions of spleens from immunized mice were prepared and mixed with SP2 / 0 cells at a 1:2 ratio. Cells were fused using polyethylene glycol and then distributed into 96-well cell culture plates. Medium was changed 7 days and 10–14 days after fusion. Wells / hybridomas were screened for reactivity to AβpE3-42 protofibrils using ELISA. Positive clones were diluted using a limiting dilution assay to confirm monoclonality. Desired clones were cryopreserved, expanded for antibody production, and sequenced.

[0104] Antigens used for hybridoma screening by ELISA: AβpE3-42 and Aβ1-42 monomers (American Peptide) and AβpE3-42 and Aβ1-42 protofibrils were used to screen hybridoma clones. Protofibrils were generated using each monomer. Briefly, to generate AβpE3-42 protofibrils, the AβpE3-42 peptide was dissolved in 10 mM NaOH, 0.005% Tween-20, pH >11 at a concentration of 100 μM. Protofibrils were prepared by neutralizing the AβpE3-42 peptide to pH 7.4 by adding 1:1 2x PBS buffer to a final concentration of 50 μM. The peptide was incubated for approximately 30 minutes at 37°C for protofibril formation and purified from remaining monomers by HPLC on a Superdex 75 Increase 3.2 / 300 size-exclusion column using a mobile phase of 1x PBS, 0.1% Tween-20, pH 7.4. The protofibril reaction was centrifuged at 16,000 x g for 5 minutes at 4°C before injection. The void peak containing AβpE3-42 protofibrils was collected, and the concentration was determined using SEC and a calibration curve of Aβ protofibril standards of known concentrations. The same procedure was used to generate Aβ1-42 protofibrils from the corresponding monomers.

[0105] Hybridoma screening by ELISA: Supernatants from generated hybridomas were characterized using ELISA using Aβ1-42 or AβpE3-42 monomer or protofibrils as capture antigens. ELISA microtiter plates were coated with polyclonal rabbit anti-Aβ42 antibody (diluted to 0.5 μg / ml in PBS, 50 μl added per well) and incubated overnight at 4°C or for 1 hour at 37°C. The plates were washed four times with 1x ELISA wash buffer, followed by the addition of 100 μl / well of Pierce blocking buffer and incubation at room temperature for 1 hour with shaking (900 rpm) to block residual binding capacity. The blocking buffer was discarded, and each antigen (diluted to 5 nM in incubation buffer, 50 μl added per well) was added, and the plates were incubated at room temperature for 1 hour with shaking (900 rpm). The plates were washed four times as above, followed by the addition of samples. Supernatant from hybridoma cell cultures was added to each well in a volume of 50 μl / well, either undiluted or diluted 1:2 with incubation buffer. Samples were incubated at room temperature for 1 hour with shaking (900 rpm). After a washing step, HRP-conjugated anti-mouse IgG antibody (diluted 1 / 5000 in incubation buffer) was added to each well (50 μl / well), and the plate was incubated at room temperature for 1 hour with shaking (900 rpm). After an additional washing step, detection was performed by adding 50 μl of room-temperature TMB to each well, followed by a 10-minute incubation at room temperature without shaking in the dark. The reaction was stopped by adding 50 μl of 2 M H2SO4, and the microtiter plate was read at a wavelength of 450 nm, preferably within 15 minutes. Positive clones that specifically bind to AβpE3-42 were further characterized using Aβ1-42, Aβ2-42, Aβ3-42, AβpE3-42, and Aβ4-42 monomer (all purchased from Anaspec) as capture antigens in ELISA according to the method described above. Regardless of antibody concentration, the supernatant was diluted 1 / 243 in ELISA, so the measured OD 450 The values ​​did not necessarily correlate with binding affinity.

[0106] Antibody concentration measurement: The antibody concentration in each hybridoma supernatant was measured using a standard sandwich ELISA. Microtiter plates were coated overnight at 4°C with an anti-mouse IgG antibody (0.5 μg / ml, 50 μl per well) that recognizes the F(ab')2 portion of mouse IgG. Residual binding capacity of the plate was blocked by adding 200 μl / well of blocking buffer and incubating at room temperature for 1 hour with shaking (900 rpm). The plate was washed three times with 1x ELISA wash buffer, followed by the addition of samples. Hybridoma supernatants were diluted 1 / 250 and added to the ELISA plate in duplicate (200 μl / well), followed by seven two-fold serial dilutions to ensure that the sample dilutions were within the standard range. A standard curve was generated using the Aβ protofibril-selective mouse antibody mAb158 (Englund et al. (2007), J Neurochem 103(1):334-45) by two-fold serial dilutions to create a final concentration range of 15–500 pg / ml. The plate was incubated at room temperature for 2 hours with shaking (900 rpm). The plate was washed as above, and 50 μl of HRP-conjugated anti-mouse IgG antibody (diluted 1 / 2500 in incubation buffer) that is also F(ab)2-specific was added to each well, followed by incubation at room temperature for 1 hour with shaking (900 rpm). For detection, the plate was washed again as above, and 100 μl of room-temperature TMB was added to each well. The plate was incubated at room temperature for 5–20 minutes without shaking. The reaction was stopped by adding 50 μl of 2 M H2SO4, and the plate was read at 450 nm within 15 minutes.

[0107] result Generation of monoclonal antibodies using hybridoma technology: Antibodies that selectively bind to AβpE3 were generated by immunization with AβpE3-42 protofibrils. Plasma samples were analyzed for reactivity against AβpE3-40 monomers by ELISA. When the titer reached at least 1 / 100,000, mice were sacrificed, and spleens were harvested and used for hybridoma generation.

[0108] A total of 22 AβpE3-42-reactive hybridomas were generated. Their specificity for AβpE3-42 monomer and AβpE3-42 protofibrils compared with AβpE3-42 monomer and AβpE3-42 protofibrils was tested by ELISA. Twelve of the clones bound to all four Aβ forms tested, while 10 of the clones specifically bound AβpE3-42. None of the clones were selective for protofibrils, but they bound AβpE3-42 monomer as well as AβpE3-42 protofibrils well.

[0109] Ten clones that were specific binders to AβpE3-42 were further characterized in ELISA using Aβ1-42, Aβ2-42, Aβ3-42, AβpE3-42, and Aβ4-42 monomer as capture reagents. All ten clones tested were specific for AβpE3-42 monomer and had only slight cross-reactivity with Aβ3-42 monomer. Figure 1 shows the results of this experiment for two selected clones, designated Pyr7.1 and Pyr12.2.

[0110] To compare the binding strength of the Pyr7.1 and Pyr12.2 antibody clones, we first determined the antibody concentrations and then loaded the same amount of each clone onto an ELISA plate using AβpE3-42 protofibrils as capture. Both clones showed binding to AβpE3-42 protofibrils, and the binding was comparable to that of the positive control mAb 6E10 (Figure 2).

[0111] Example 2 Hybridoma sequencing and recombinant antibody production Materials and Methods Hybridoma Sequencing: Hybridoma clones producing monoclonal antibodies with demonstrated specificity for AβpE3-42 monomers and protofibrils, generated and characterized as described in Example 1, were sequenced by whole-transcriptome shotgun sequencing. The DNA and protein sequences of the mature VH and VL regions were identified.

[0112] Expression, production, and purification: Prior to synthesis, the variable domains were designed and optimized for expression in mammalian cells (HEK293). The sequences were then subcloned into cloning expression vectors (Absolute Antibody) for the appropriate immunoglobulin heavy and light chain isotypes and subtypes. HEK293 cells were passaged to a stage optimal for transient transfection. Cells were transiently transfected with heavy and light chain expression vectors and cultured for an additional 6–14 days. The cultures were harvested and single-step purification was performed using affinity chromatography, after which the purified antibody was buffer-exchanged into PBS. The antibody was analyzed for purity by SDS-PAGE, and concentration was determined by UV spectroscopy.

[0113] result Hybridoma sequencing and recombinant antibody generation: Hybridoma clones with demonstrated specificity for AβpE3-42 monomers and protofibrils were sequenced, and the sequences of selected antibodies Pyr7.1 and Pyr12.2 are reported.

[0114] The amino acid sequences of the entire antibodies were obtained. The amino acid sequences of each variable heavy (VH) and variable light (VL) chain, as well as the sequence of the constant region shared by all antibodies, are shown in Table 1 below. Complementarity-determining regions (CDRs) were identified using the Kabat definition.

[0115] [Table 1]

[0116] [Table 2]

[0117] Monoclonal antibodies Pyr7.1 and Pyr12.2 were selected for production as recombinant IgG2c antibodies. All recombinant antibodies were successfully produced and purified to a final concentration of 1 mg / ml. Antibody purity, as defined by SEC-HPLC, was greater than 98% monomer for all antibodies.

[0118] Example 3 Characterization of recombinant antibodies This example describes the characterization of the affinity, selectivity, and specificity of the recombinant antibodies produced in Example 2 by inhibition ELISA and SPR.

[0119] Materials and Methods Aβ monomer species and Aβ protofibrils: To characterize the binding of recombinant antibodies to different Aβ species, the following Aβ peptides were used: AβpE3-40, Aβ1-40, Aβ1-28, and AβpE11-40 (all purchased from Bachem), and Aβ2-28, Aβ3-28, Aβ4-28, and AβpE11-28 (all custom-made by Innovagen and purchased from Innovagen). Aβ peptides obtained from Bachem were dissolved at a concentration of 100 μM in 10 mM NaOH, 0.005% Tween-20, pH >11. Aβ peptides obtained from Innovagen were dissolved at a concentration of 300 μM in 1× PBS, pH 7.4. Aliquots were prepared and stored at -80°C until analysis. All peptides were confirmed to be monomeric by size-exclusion chromatography.

[0120] Protofibrils were prepared using AβpE3-42 peptide from Bachem. Briefly, AβpE3-42 peptide was dissolved at a concentration of 100 μM in 10 mM NaOH, 0.005% Tween-20, pH >11. Protofibrils were prepared by neutralizing the AβpE3-42 peptide to pH 7.4 by adding 1:1 2x PBS buffer to a final concentration of 50 μM. The peptide was incubated at 37°C for approximately 30 minutes for protofibril formation and purified from residual monomers by HPLC on a Superdex 75 Increase 3.2 / 300 size-exclusion column using a mobile phase of 1x PBS, 0.1% Tween-20, pH 7.4. Prior to injection, the protofibril reaction was centrifuged at 16,000 × g for 5 minutes at 4°C to remove insoluble fibrils. The void peak containing AβpE3-42 protofibrils was collected and the concentration determined using SEC and a calibration curve of Aβ protofibril standards with known concentrations.

[0121] Selectivity evaluation by inhibition ELISA and IC 50 Determination: The binding of the selected recombinant antibodies Pyr7.1 and Pyr12.2 to different Aβ antigens (AβpE3-40, Aβ1-40, and AβpE11-40 monomers and AβpE3-42 protofibrils) was assessed by inhibition ELISA. For binding to Aβ1-40 and AβpE11-40, a positive control, 4G8 (Covance #SIG-39320, epitope: amino acids 17-24 of Aβ), was included. The recombinant antibodies were incubated at a fixed concentration (0.05 μg / ml) with varying concentrations of Aβ antigens. After 45 minutes of incubation at 900 rpm to reach equilibrium, the antibody-Aβ samples were added to ELISA plates that had been blocked and washed with AβpE3-40 (0.5 μM). Samples were incubated on the plate without shaking for 25 minutes, followed by washing, incubation with detection antibody, another washing step, and finally incubation with alkaline phosphatase substrate. Optical density readings at 405 nm were taken, and the collected data were analyzed using a four-parameter variable slope nonlinear fit to determine the IC. 50value was determined.

[0122] Evaluation of affinity and specificity by surface plasmon resonance and K D Determination: Binding interactions between antigens and antibodies were assessed by SPR using a Biacore 8K instrument (Cytiva) according to standard procedures. The binding of selected recombinant antibodies Pyr7.1 and Pyr12.2 to AβpE3-40 monomers and protofibrils, as well as their selectivity for Aβ1-28 monomers, were assessed. Furthermore, specificity for different N-truncated forms of Aβ (Aβ2-28, Aβ3-28, Aβ4-28, and AβpE11-28 monomers) was assessed.

[0123] Single-cycle kinetics with antibody immobilized on a CM5 chip was used to measure antibody binding to different monomers (AβpE3-40, AβpE11-28, Aβ1-28, Aβ2-28, Aβ3-28, and Aβ4-28). For measurements, 5 μg / ml of analyte antibody was immobilized on the chip. AβpE3-40 monomers were then injected over the chip using five 2-fold dilutions starting at 250 nM for AβpE3-40 and a 20-minute dissociation time, and 2500 nM for all other monomers and a 10-minute dissociation time. Regeneration of the surface between cycles was performed by injecting 30 μl of 3 M MgCl2. Binding data were fitted to a 1:1 interaction model.

[0124] Antibody binding to AβpE3-42 protofibrils was also measured using single-cycle kinetics. AβpE3-42 protofibrils (138 ng / ml) were coupled to a CM5 chip using standard Biacore coupling chemistry (low immobilization level). For binding to AβpE3-42 protofibrils, the antibody was injected over the chip using a 3-fold dilution series in five steps starting at 700 nM, with a 2-minute injection of each antibody concentration and a 60-minute dissociation time. Regeneration of the surface between cycles was performed by injecting 30 μl 10 mM glycine-HCl pH 1.7. Binding data were fitted to a 1:1 interaction model. Using the 1:1 interaction model, the K for antibody binding to Aβ monomers was calculated. D and the apparent K of antibody binding to Aβ protofibrils. D is obtained.

[0125] In all SPR experiments, 1x HBS-EP+ (Cytiva, Cat. No. BR100669) was used to dilute antibodies and target antigens. Experiments were performed at 25°C.

[0126] result Selectivity evaluation by inhibition ELISA and IC 50 Determination of IC: The binding of recombinant antibodies to AβpE3-40 and their selectivity for AβpE11-40 and AβpE11-40 monomers was first assessed using an inhibition ELISA. Recombinant antibodies Pyr7.1 and Pyr12.2 showed binding to dissolved AβpE3-40 monomers (Figure 3). None of the antibodies bound to dissolved AβpE3-40 at concentrations up to 5 μM, indicating an IC 50 This suggests that IC values ​​were above 5 μM (Figure 4). The positive control antibody 4G8 showed binding to Aβ1-40, as expected. The recombinant antibodies Pyr7.1 and Pyr12.2 were also tested for binding to AβpE11-40 monomers using an inhibition ELISA. Here, no binding was observed at antigen concentrations up to 5 μM, with IC 50 The calculated IC values ​​were greater than 5 μM (Figure 5). The positive control antibody 4G8 showed binding to AβpE11-40, as expected.50 The values ​​are listed in Table 2.

[0127] The binding of the recombinant antibodies to AβpE3-42 protofibrils was assessed using an inhibition ELISA. Both antibodies showed binding to dissolved AβpE3-42 protofibrils (Figure 6). The calculated IC 50 The values ​​are listed in Table 2.

[0128] [Table 3]

[0129] Evaluation of affinity and specificity by surface plasmon resonance and K D Determination: Recombinant antibodies Pyr7.1 and Pyr12.2 were evaluated for affinity and specificity by SPR and their K D value was determined.

[0130] Both antibodies showed binding to AβpE3-40 monomers and AβpE3-42 protofibrils. d The apparent affinities of Pyr7.1 and Pyr12.2 for AβpE3-42 protofibrils were likely underestimated because the values ​​were outside the detection limits of the instrument. All kinetic data from such binding experiments were excluded when calculating affinities. The calculated k a , k d and (apparent) K D The values ​​are shown below in Tables 3 and 4. Representative sensorgrams are shown in Figures 7 and 8.

[0131] [Table 4]

[0132] [Table 5]

[0133] The recombinant antibody showed no binding to monomeric Aβ1-28 up to 2500 nM.

[0134] Specificity of recombinant antibodies assessed by surface plasmon resonance: The specificity of the recombinant antibodies for different N-truncated forms of Aβ monomer was assessed by SPR. Pyr7.1 and Pyr12.2 did not bind to Aβ2-28 monomer over the concentration range tested. Pyr12.2 bound to Aβ3-28 monomer with nM affinity, while no binding was detected for Pyr7.1. Both antibodies bound to Aβ4-28 monomer with μM affinity. No binding was observed for either antibody against AβpE11-28 up to a concentration of 2500 nM. The calculated k a , k d and K. D The values ​​are shown in Table 5.

[0135] [Table 6]

[0136] Example 4 Target binding of recombinant antibodies in brains from human Alzheimer's disease patients and non-demented controls This example describes the target binding of recombinant antibodies Pyr7.1 and Pyr12.2, produced as described in Example 2, tested by immunoprecipitation on human brain extracts from AD patients and NDE controls and by immunohistochemistry on human brain sections.

[0137] Materials and Methods Brain tissue homogenization and sample preparation: Fresh-frozen human brain cortical tissue from Alzheimer's disease (AD) patients and non-demented (NDE) controls was homogenized in a Potter-Elvehjem homogenizer at a 1:10 weight:volume ratio in Tris-buffered saline (TBS) buffer, followed by centrifugation at 16,000 × g for 1 hour. The resulting supernatant was frozen at −80°C until analysis.

[0138] Target binding in human Alzheimer's disease brain extracts by immunoprecipitation: Antibody binding to targets in human AD brains was analyzed by immunodepletion, a method that uses antibodies specific for the target molecule to remove target proteins from samples. Briefly, recombinant antibodies Pyr7.1 and Pyr12.2, covalently coupled to magnetic Dynabeads, were incubated with soluble TBS brain extracts from AD and non-demented (NDE) control cases for 1 hour at room temperature with rotation. The bead-bound target was separated by a magnet and depleted from the extracts. The depleted brain extracts (supernatants) were analyzed using an AβpE3-40 kit and MSD assay for measurement of AβpE3-42 levels. Target binding by the recombinant antibodies was assessed by a reduction in the measured AβpE3-40 or AβpE3-42 levels in the depleted samples compared to those in the undepleted brain extracts. Complete target binding was demonstrated when the levels in the depleted brain extracts were below the lower limit of quantitation (LLOQ) of the assay.

[0139] A sandwich ELISA kit (product code: 27418) from Immunobiological Laboratories was used to measure AβpE3-40 levels in human AD brains. Each assay kit contains all necessary components, including antibodies, standard calibrators, and plates pre-coated with a human anti-Aβ mouse IgG monoclonal capture antibody (epitope at amino acids 35–40 of Aβ). Briefly, diluted standard calibrators and test samples were bound to the plate during overnight incubation at 4°C. After a washing step, an HRP-conjugated anti-human AβpE3 antibody (8E1, included in the kit) was added, followed by a 1-hour incubation at 4°C. After an additional washing step, TMB was added as a coloring agent (chromogen), and the plate was incubated for 30 minutes before the reaction was stopped and the absorbance was measured at 450 nm. The intensity of the color was proportional to the amount of human AβpE3-40.

[0140] AβpE3-42 was measured using the MSD assay. MSD standard plates were coated overnight at 4°C with Pyr7.1 (3 μg / ml per well) as a capture antibody. Free binding sites were blocked by incubation with 1% Blocker A solution, followed by 2-hour incubation (shaking at 900 rpm) with diluted standards (AβpE3-42, 7.1–1000 pg / ml) and test samples. Anti-Aβ42 rabbit polyclonal antibody (in-house production) was added to the plate (1.5 μg / ml per well) and incubated for 1 hour, followed by a final 1-hour incubation with goat anti-rabbit MDS SULFO-TAG antibody (diluted 1:1000). The plate was washed between blocking and each antibody incubation step. The plate was read on an MSD Sector Imager, where a light signal was generated and measured. The signal intensity correlated with the amount of AβpE3-42 in the sample.

[0141] Target binding in human Alzheimer's disease brains by immunohistochemistry: Immunohistochemistry (IHC) analysis was performed on brain tissue from AD and non-demented controls. Postmortem human brain tissue from the temporal cortex was obtained from the Netherlands Brain Bank (NBB) and collected at autopsy with the approval of the local ethical committee.

[0142] Mouse anti-human Aβ antibodies 6E10 (Covance #SIG-39320) and 4G8 (Covance #SIG-39200) were used to detect Aβ pathology in brain sections. Purified mouse monoclonal anti-AβpE3 IgG1 antibody (Glu3) was used as a reference antibody (Biolegend, #822301). Human target binding in AD brains was evaluated for antibodies Pyr7.1 and Pyr12.2 obtained as described in the above examples.

[0143] For IHC staining of Aβ, we used an automated staining robot and an HRP-3,3'-diaminobenzidine (DAB)-based detection system (Discovery XT and OmniMap DAB kit, Ventana Medical Systems). IHC analysis was performed on a subset of formalin-fixed, paraffin-embedded tissue sections and fresh-frozen tissue sections. All tissues were cut into 4-8 μm-thick sections and mounted on Superfrost Plus slides (Thermo Fisher). For fresh-frozen brain samples, tissues were sectioned onto Superfrost Plus slides, air-dried for 30 min, and transferred directly to ice-cold 50% acetone for 30 s, followed by 100% acetone for 5 min, and finally to 1x PBS for 5 min before wet-loading in the Ventana robotic platform. The working concentrations used for Pyr7.1 and Pyr12.2 were 1 μg / ml, and the reference antibody Glu3 was used at 0.5 μg / ml. Visualization of the primary / secondary antibody complex was performed by adding hydrogen peroxide and DAB, which resulted in an insoluble brown-stained precipitate at the antibody binding site. Counterstaining was performed with hematoxylin (HTX). Stained slides were scanned in bright field using a Pannoramic 250 FLASH II slide scanner. The resulting image files were uploaded to viewer software (Pannoramic Viewer) and adjusted for optimal brightness and contrast for manual evaluation of the staining results.

[0144] result Target binding in human Alzheimer's disease brain extracts by immunoprecipitation: The recombinant antibodies Pyr7.1 and Pyr12.2 were tested for their ability to selectively bind to dissolved AβpE3-40 and AβpE3-42 in human brain extracts from AD patients. Immunoprecipitation (IP) of TBS brain extracts from AD patients using the recombinant antibodies demonstrated depletion of AβpE3-40 (Figure 9) and AβpE3-42 (Figure 10) levels by both antibodies. No measurable levels of AβpE3-40 or AβpE3-42 could be detected in brain TBS extracts from NDE control cases.

[0145] Target binding in human Alzheimer's disease brains by immunohistochemistry: Immunohistochemical staining of brain sections from AD individuals (confirmed to have Aβ pathology by IHC staining with 6E10 / 4G8, not shown) with recombinant antibodies Pyr7.1 and Pyr12.2 resulted in specific binding by both antibodies to nuclear and diffuse plaques in AD brains, with identical staining patterns. No binding was observed in NDE control brains. Representative images from immunostaining with Pyr7.1 and Pyr12.2 on adjacent sections from formalin-fixed, paraffin-embedded AD or NDE control brains are shown in Figure 11.

[0146] Example 5 Humanization of Pyr12.2 This example describes the humanization of the murine AβpE3-specific antibody Pyr12.2 described in Examples 2-4, and the subsequent generation of humanized Pyr12.2 variants.

[0147] Materials and Methods Humanization: Pyr12.2 was humanized by grafting the CDRs into IGHV1-46*01 and IKKV2-30*02 human variable domains and making different backmutations to murine residues at various positions. Additional beneficial mutations were made in the framework regions of one of the variants.

[0148] Expression of each variant from transient transfection: Humanized antibodies were expressed in CHO cells and purified by affinity chromatography, followed by buffer exchange into phosphate-buffered saline (PBS). Purified antibodies were characterized using SDS-PAGE, SEC, and UV protein measurement.

[0149] result Antibody humanization and generation: The sequences of the humanized VH and VL and the common human constant regions of the heavy and light chains are shown in Table 6 for two humanized Pyr12.2 variants, designated H2L7 and H9L8.

[0150] [Table 7]

[0151] [Table 8]

[0152] Expression and purification of the humanized antibody variants was performed as described in Example 2 for the recombinant murine antibody.

[0153] Example 6 Characterization of affinity, selectivity, and specificity of humanized antibodies This example describes the characterization of the affinity, selectivity, and specificity of the humanized Pyr12.2 mutant antibodies H2L7 and H9L8 generated and produced in Example 5 by inhibition ELISA and SPR.

[0154] Materials and Methods Aβ monomer species and Aβ protofibrils: The following Aβ peptides were used to characterize the binding of humanized antibodies to different Aβ species: AβpE3-40, Aβ1-40, Aβ1-28, and AβpE11-40 (all purchased from Bachem), and Aβ2-28, Aβ3-28, Aβ4-28, Aβ5-28, and AβpE11-28 (all custom-made by Innovagen and purchased from Innovagen). Aβ peptides were dissolved at a concentration of 100 μM in 10 mM NaOH, 0.005% Tween-20, pH >11. Aliquots were made and stored at -80°C until analysis. All peptides were confirmed to be monomeric by size-exclusion chromatography.

[0155] Protofibrils were prepared using AβpE3-42 peptide from Bachem. Briefly, AβpE3-42 peptide was dissolved at a concentration of 100 μM in 10 mM NaOH, 0.005% Tween-20, pH >11. Protofibrils were prepared by neutralizing AβpE3-42 peptide to pH 7.4 by adding 1:1 2x PBS buffer to a final concentration of 50 μM. The peptide was incubated at 37°C for approximately 30 minutes for protofibril formation and purified from remaining monomers by HPLC on a Superdex 75 Increase 3.2 / 300 size-exclusion column using a mobile phase of 1x PBS, 0.1% Tween-20, pH 7.4. Prior to injection, the protofibril reaction was centrifuged at 16,000 × g for 5 minutes at 4°C to remove insoluble fibrils. The void peak containing AβpE3-42 protofibrils was collected, and the concentration was determined using SEC and a calibration curve of Aβ protofibril standards with known concentrations. Using the same procedure, Aβ1-42 protofibrils were generated from the corresponding Aβ1-42 monomers (Bachem).

[0156] Specificity assessment by inhibition ELISA and IC 50 Determination of IC: The specificity of the humanized antibodies H2L7 and H9L8 for AβpE3-28, compared with N-terminally intact Aβ (Aβ1-28) and different N-truncated forms of Aβ (Aβ2-28, Aβ3-28, Aβ4-28, Aβ5-28, and AβpE11-28 monomer), was assessed by inhibition ELISA. The humanized antibodies were incubated at a fixed concentration (0.5 μg / ml) with adjusting concentrations of various Aβ antigens. After 45 minutes of incubation at 900 rpm to reach equilibrium, the antibody-Aβ samples were added to ELISA plates that had been blocked and washed with AβpE3-40 (0.5 μM). The samples were incubated on the plate for 25 minutes without shaking, followed by washing, incubation with the detection antibody, another wash step, and finally incubation with alkaline phosphatase substrate. Optical density readings at 405 nm were taken, and the collected data were analyzed using a four-parameter variable slope nonlinear fit to determine IC. 50value was determined.

[0157] Selectivity evaluation by inhibition ELISA and IC 50 Determination of IC Binding of humanized antibodies H2L7 and H9L8 to AβpE3-40 and AβpE3-42 protofibrils, as well as their selectivity for Aβ1-40 monomers and Aβ1-42 protofibrils, was assessed by inhibition ELISA. Humanized antibodies were incubated at a fixed concentration (0.1 μg / ml) with various Aβ antigens at adjustable concentrations. After 45 minutes of incubation at 900 rpm to reach equilibrium, antibody-Aβ samples were added to ELISA plates that had been blocked and washed with AβpE3-40 (0.5 μM). Samples were incubated on the plate for 25 minutes without shaking, followed by washing, incubation with detection antibody, another wash step, and finally incubation with alkaline phosphatase substrate. Optical density readings at 405 nm were taken, and the collected data were analyzed using a four-parameter, variable-slope nonlinear fit to determine IC . 50 value was determined.

[0158] Affinity evaluation by surface plasmon resonance and K D Determination: Binding interactions between antigen and antibody were assessed by SPR using a Biacore 8K instrument (Cytiva) according to standard procedures. The binding of humanized antibodies H2L7 and H9L8 to AβpE3-40 monomers and AβpE3-42 protofibrils was assessed.

[0159] Binding of antibodies to AβpE3-40 monomers was measured using single-cycle kinetics with antibodies immobilized on a CM5 chip. For measurements, 5 μg / ml of analyte antibody was immobilized on the chip. The monomer was then injected over the chip using five 2-fold dilutions starting from 250 nM, with a 2-minute injection time for each antibody concentration and a 20-minute dissociation time. Regeneration of the surface between cycles was performed by injecting 30 μl of 3 M MgCl2. Binding data were fitted to a 1:1 interaction model.

[0160] Single-cycle kinetics was used to measure antibody binding to AβpE3-42 protofibrils. AβpE3-42 protofibrils (138 ng / ml) were coupled to a CM5 chip using standard Biacore coupling chemistry (low immobilization level). For binding to AβpE3-42 protofibrils, antibodies were injected over the chip using a five-step, four-fold dilution series starting at 150 nM, with a 2-minute injection time for each antibody concentration and a 20-minute dissociation time. Regeneration of the surface between cycles was performed by injecting 30 μl 10 mM glycine-HCl pH 1.7. Binding data were fitted to a 1:1 interaction model. Using the 1:1 interaction model, the K for antibody binding to Aβ monomers was calculated. D and the apparent K for antibody binding to Aβ protofibrils D is obtained.

[0161] In all SPR experiments, 1x HBS-EP+ (Cytiva, Cat. No. BR100669) was used to dilute antibodies and target antigens. Experiments were performed at 25°C.

[0162] result Specificity assessment by inhibition ELISA and IC 50 Determination of specificity: The specificity of humanized antibodies H2L7 and H9L8 for AβpE3-28 compared to N-terminally intact Aβ (Aβ1-28) and different N-truncated forms of Aβ (Aβ2-28, Aβ3-28, Aβ4-28, Aβ5-28, and AβpE11-28 monomer) was assessed by inhibition ELISA. Humanized antibodies H2L7 and H9L8 showed the highest binding to dissolved AβpE3-28 monomer and some cross-reactivity to Aβ3-28 (Figure 12). None of the antibodies bound to dissolved Aβ1-28, Aβ2-28, Aβ4-28, Aβ5-28, or AβpE11-28 monomer at concentrations up to 12.5 μM, indicating an IC 50 This suggests that the calculated IC values ​​were greater than 12.5 μM for these antibodies. 50 The values ​​are listed in Table 7.

[0163] [Table 9]

[0164] Selectivity evaluation by inhibition ELISA and IC 50 Determination of IC Binding of humanized antibodies H2L7 and H9L8 to AβpE3-40 monomers and AβpE3-42 protofibrils, as well as selectivity for Aβ1-40 monomers and Aβ1-42 protofibrils, was assessed using an inhibition ELISA. Humanized antibodies H2L7 and H9L8 demonstrated binding to dissolved AβpE3-40 monomers and AβpE3-42 protofibrils ( FIG. 13 ). Neither antibody bound to dissolved Aβ1-40 monomers at concentrations up to 12.5 μM, indicating that IC for these antibodies was not determined. 50 None of the antibodies bound to dissolved Aβ1-42 protofibrils at concentrations up to 500 nM, suggesting that the IC 50 This suggests that the calculated IC 50 The values ​​are listed in Table 8.

[0165] [Table 10]

[0166] Affinity evaluation by surface plasmon resonance and K D Determination: The binding of humanized antibodies H2L7 and H9L8 to AβpE3-40 monomers and AβpE3-42 protofibrils was assessed by SPR, and their K D value was determined.

[0167] Both antibodies showed binding to AβpE3-40 monomers and AβpE3-42 protofibrils. a , k d and (apparent) K D The values ​​are shown below in Tables 9 and 10. Representative sensorgrams are shown in Figures 14 and 15.

[0168] [Table 11]

[0169] [Table 12]

[0170] Example 7 Target Binding of Humanized Antibodies in Brains from Human Alzheimer's Disease Patients and Non-Demented Controls This example describes the target binding of humanized antibodies H2L7 and H9L8, produced as described in Example 5, and tested by immunoprecipitation on human brain extracts from AD patients and NDE controls and immunohistochemistry on human brain sections.

[0171] Materials and Methods Target binding in human Alzheimer's disease brain extracts by immunoprecipitation: Antibody binding to targets in human AD brains was analyzed by immunoprecipitation, a method that uses antibodies specific for the target molecule to remove target proteins in samples. Briefly, each humanized antibody was incubated with magnetic Protein A Dynabeads and soluble 16,000 × g TBS brain extract from an AD case. The TBS brain extract was prepared as described in Example 4. The bead-bound target was separated by a magnet and eluted from the beads using 70% formic acid. After neutralization, the pellet (IP fraction) was analyzed for total AβpE3-x levels using an in-house developed MSD assay. Briefly, MSD GOLD 96-well small spot streptavidin plates were coated with biotinylated Pyr12.2 antibody for 1 hour at room temperature. After a washing step, free binding sites were blocked by incubation with Diluent 35. The plate was washed again and incubated for another 2 h (shaking at 900 rpm) with a dilution series of the standard (AβpE3-40 monomer, 3.125–400 pg / ml) and the test samples. After another washing step, the conjugated detection antibody anti-Aβ4G8 SULFO tag was added to the plate at a concentration of 1 μg / ml for 1 h. Finally, the plate was washed and read using an MSD Sector Imager (S 600MM, MSD). The resulting signal correlated with the amount of AβpE3-x in the sample.

[0172] Target binding in human Alzheimer's disease brains by immunohistochemistry: Immunohistochemistry (IHC) analysis was performed on brain tissue from AD patients. Postmortem human brain tissue from the temporal cortex was obtained from the Netherlands Brain Bank (NBB) and collected at autopsy with the approval of the local ethical committee.

[0173] Mouse anti-human Aβ antibodies 6E10 (Covance #SIG-39320) and 4G8 (Covance #SIG-39200) were used to detect Aβ pathology in brain sections. Human target binding in AD brains was assessed for humanized antibodies H2L7 and H9L8.

[0174] For IHC staining of Aβ, we used an automated staining robot and an HRP-3,3'-diaminobenzidine (DAB)-based detection system (Discovery XT and OmniMap DAB kit, Ventana Medical Systems). IHC analysis was performed on fresh-frozen tissue sections. All tissues were sectioned into 4-8 μm sections and mounted on Superfrost Plus slides (ThermoFisher). Sections were air-dried for 30 min and transferred directly to ice-cold 50% acetone for 30 s, followed by 100% acetone for 5 min, and finally to 1x PBS for 5 min before wet-loading onto the Ventana robotic platform. The working concentrations used for the tested antibodies H2L7 and H9L8 and the reference antibodies 6E10 and 4G8 were 1 μg / ml. Visualization of the primary / secondary antibody complex was performed by the addition of hydrogen peroxide and DAB, which resulted in the formation of an insoluble brown-stained precipitate at the antibody binding sites. Counterstaining was performed with hematoxylin (HTX). Stained slides were scanned in bright field using a Pannoramic 250 FLASH II slide scanner. The resulting image files were uploaded to viewer software (Pannoramic Viewer) and adjusted for optimal brightness and contrast for manual evaluation of the staining results.

[0175] result Target binding in human Alzheimer's disease brain extracts by immunoprecipitation: Humanized antibodies H2L7 and H9L8 were tested for their ability to bind to dissolved AβpE3 in brain extracts from human AD patients. Immunoprecipitation (IP) of TBS brain extracts from AD patients using the humanized antibodies showed concentration-dependent IP of AβpE3-x by both antibodies (Figure 16).

[0176] Target binding in human Alzheimer's disease brains by immunohistochemistry: Immunohistochemical staining of brain sections from AD individuals (confirmed to have Aβ pathology by IHC staining with 6E10 / 4G8) with humanized antibodies H2L7 and H9L8 resulted in specific binding by both antibodies to nuclear and diffuse plaques in AD brains, with identical staining patterns. No binding was observed in NDE control brains (data not shown). Representative images from immunostaining with H2L7 and H9L8 on adjacent sections from fresh-frozen AD brains are shown in Figure 17.

[0177] Example 8 Characterization of the functional effects of humanized antibodies This example describes the functional effects of the humanized AβpE3 antibodies H2L7 and H9L8 generated and produced in Example 5. The ability of the humanized antibodies to inhibit AβpE3 aggregation and clear amyloid plaques ex vivo in AD brain slices was assessed.

[0178] Materials and Methods Inhibition of AβpE3-42 aggregation: The effect of humanized antibodies on the aggregation of AβpE3-42 monomers was assessed in an aggregation assay using thioflavin T (ThT, Sigma T3516). AβpE3-42-NH4+ monomers (Bachem, H4916, 2 μM) were mixed with ThT (5 μM) and either H2L7 or H9L8 humanized antibodies (25–800 nM) or an IgG1 isotype control antibody (CrownVivo, C-00012, 800 nM) in phosphate-buffered saline (PBS) (pH 8.2), 200 mM NaCl, and 10 μM EDTA in a 384-well plate (Thermo Scientific #242764) on ice. The plate was then transferred to a Tecan SPARK intensified plate reader equipped with a 448 ± 7 nm excitation filter and a 485 ± 20 nm emission filter at 37 °C to record ThT fluorescence over a period of 36-72 h. Data were background corrected and maximum fluorescence was plotted against antibody concentration to determine IC. 50 was calculated. The experiment was repeated 3 to 5 times. Statistical significance was tested by two-way ANOVA.

[0179] Ex vivo phagocytosis in AD brain: An ex vivo phagocytosis assay was used to investigate whether humanized antibodies could induce plaque clearance by macrophages. Fresh-frozen AD brain tissue was cryosectioned (20 μm), and sections were collected on 12 mm glass coverslips coated with poly-D-lysine (Gibco A38904-01, 50 μg / ml). Next, sections were incubated with humanized antibodies (1 μg / ml) or an IgG1 isotype control antibody (CrownVivo, C-00012, 1 μg / ml) for 1 hour at 37°C, 5% CO2. Sections were then washed once and 5 × 10 IgG1 isotype control antibodies isolated from the buffy coat were added. 5 ~1×10 6 The cells were incubated with primary human macrophages for 24 hours. Plaque clearance was assessed by measuring the immunopositive area of ​​each section after immunohistochemistry with mouse anti-human Aβ antibodies 6E10 (Covance #SIG-39320) and 4G8 (Covance #SIG-39200). Experiments were repeated two to five times using macrophages isolated from different buffy coats. Statistical significance was tested by one-way ANOVA.

[0180] result Inhibition of AβpE3-42 aggregation: The ability of humanized antibodies H2L7 and H9L8 to inhibit AβpE3-42 aggregation was assessed. Both H2L7 and H9L8 inhibited AβpE3-42 fibril formation in a concentration-dependent manner, as demonstrated by a decrease in the maximum ThT fluorescence signal (Fmax) in the presence of the antibodies (Figure 18).

[0181] Ex vivo phagocytosis in AD brain: The ability of humanized antibodies H2L7 and H9L8 to induce clearance of Aβ plaques by macrophages in AD brains was evaluated. Compared to negative control samples preincubated with no antibody or an isotype control IgG1 antibody, Aβ plaques were significantly reduced after preincubation with H2L7 or H9L8. The results show that both antibodies can induce plaque clearance by macrophages (Figure 19).

[0182] Example 9 Pharmacokinetic profiles of murine and humanized AβpE3 antibodies This example describes the pharmacokinetic (PK) profiles in mice after administration of the murine antibody Pyr12.2, generated and produced as described in Example 2, or the humanized antibodies H2L7 and H9L8, generated and produced as described in Example 5.

[0183] Materials and Methods Antibody administration and sample collection: Each antibody was administered intravenously (iv) via the tail vein to 8-week-old female C57BL / 6J mice (5 mice per antibody) at a dose of 10 mg / kg. Blood samples were collected 5 minutes, 4 hours, 24 hours, 72 hours, 168 hours (7 days), 336 hours (14 days), 672 hours (28 days), and 840 hours (35 days) after the intravenous injection. Blood was collected into Microvette EDTA tubes and placed on regular ice immediately after collection. Immediately after collection (within 30 minutes), samples were centrifuged at 2400 × g for 10 minutes at +4°C. Plasma was collected and stored at -80°C until biological analysis.

[0184] Determination of antibody concentrations in plasma: The concentration of Pyr12.2 in EDTA plasma samples was determined using an MSD-based method. Briefly, MSD standard 96-well plates were coated with 0.5 μM monomeric AβpE3-40 (Bachem) overnight at 4°C. Free binding sites were blocked by incubation with 1% Blocker A (MSD Blocker A in 1x PBS-Tween 20) for 1 hour at room temperature with shaking. Washes were performed before blocking and before each subsequent incubation step. Standards and plasma samples were added and incubated for 2 hours at room temperature with shaking. Bound antibody was detected by incubation with MSD SULFO-TAG-labeled goat anti-mouse IgG antibody (R32AC-1, 0.5 μg / ml) for 1 hour at room temperature with shaking. Read buffer T (MSD 2x) was added, and the plate was read using an MSD sector imager. Signal intensity correlated with the amount of Pyr12.2 in the sample.

[0185] A commercially available kit from MSD (catalog number: K150JLD) for measuring human / NHP IgG was used to determine the concentration levels of H2L7 and H9L8 in EDTA plasma samples. Standards and plasma samples were added to a pre-coated anti-human IgG MSD plate and incubated for 2 hours at room temperature with shaking. After a wash step, anti-human / NHP IgG antibody conjugated with a SULFO-TAG label was added and incubated for 2 hours. After a wash step, MSD read buffer was added. Upon reading with an MSD sector imager, a light signal was generated and measured. The signal intensity correlated with the amount of H2L7 or H9L8 in the sample.

[0186] Pharmacokinetic Analysis: Noncompartmental analysis (NCA) of plasma concentration data was performed using Phoenix WinNonlin software. Individual observed plasma concentration versus time profiles were subjected to PK evaluation. Nominal doses and time points were used for the analysis. The maximum concentration, Cmax, was derived directly from the observed concentration versus time curve. The calculation method in NCA was set to linear up log down, with the linear trapezoidal rule used for increasing concentration versus time data and the logarithmic trapezoidal rule used for decreasing concentration data. Calculated parameters included the area under the concentration versus time curve (AUC) to the last observation time point (AUClast) or to infinity (AUCinf), calculated as AUClast + Ct / λz, where Ct is the last observation time point and λz is the elimination rate constant estimated using log-linear regression during the terminal elimination phase. The terminal half-life (t1 / 2) was calculated as ln(2) / λz, and clearance (CL) was calculated as dose / AUCinf.

[0187] result The plasma PK profiles of the murine Pyr12.2 antibody and the humanized antibodies H2L7 and H9L8 were evaluated after a single intravenous bolus injection into C57BL / 6 mice. The plasma concentration profile as a function of time is shown in Figure 20, and the calculated PK parameters are shown in Table 11. H2L7 exhibited a plasma PK profile similar to that of the murine antibody Pyr12.2 in mice, whereas the H9L8 variant surprisingly exhibited a better plasma PK profile with a longer half-life, higher total exposure (AUC), and lower clearance (CL).

[0188] [Table 13]

[0189] Example 10 Generation of humanized antibodies with improved pharmacokinetic profiles This example describes the generation of variants of the humanized antibody H2L7 with improved pharmacokinetic (PK) profiles.

[0190] Materials and Methods Design and Expression of H2L7 Mutants: To identify positively charged amino acids that could potentially cause differences in the PK profiles between the humanized antibodies H2L7 and H9L8, the HC and LC sequences of the humanized antibodies H2L7 and H9L8 from the previous example were compared (Table 11). The sequences were also compared to the murine parent antibody Pyr12.2. The structures of H2L7 and H9L8 were modeled in silico, and surface analysis was performed. Amino acids in H2L7 identified as contributing to positively charged patches were mutated to neutral amino acids. Various mutations were combined to generate several new antibodies. New surface analysis was performed on the mutant antibodies. A total of six antibodies with mutations that reduced the positively charged patches in the surface analysis were expressed as described in Example 5 for H2L7 and H9L8 and purified by an additional preparative-grade SEC step.

[0191] Antibody administration and sampling: Antibody variants were administered intravenously via the tail vein to 8-week-old female C57BL / 6J mice (5 mice per antibody) at a dose of 10 mg / kg. Blood samples were collected 5 minutes, 4 hours, 24 hours, 72 hours, 168 hours (7 days), 336 hours (14 days), 672 hours (28 days), and 840 hours (35 days) after intravenous injection. Blood was collected into Microvette EDTA tubes and placed on regular ice immediately after collection. Immediately after collection (within 30 minutes), samples were centrifuged at 2400 x g for 10 minutes at 4°C. Plasma was collected and stored at -80°C until bioanalysis.

[0192] Determination of antibody concentrations in plasma: Concentrations of antibody variants were determined in EDTA plasma samples using a commercially available kit from MSD (catalog number: K150JLD) for measuring human / NHP IgG as described in Example 9 for H2L7 and H9L8.

[0193] Pharmacokinetic Analysis: The individual observed profiles of plasma concentration versus time were subjected to PK evaluation as described in Example 9 for H2L7 and H9L8.

[0194] result Design of H2L7 variants with improved pharmacokinetic profiles: From sequence alignment of VH2 (SEQ ID NO: 22) and VH9 (SEQ ID NO: 21), residues R71 and R83 found in VH2 were highly likely to contribute to the higher clearance of H2L7 compared to VH9 (including A71 and T83) in H9L8. K12 in VH2 was also considered a candidate for mutation because the corresponding residue at this position in the parent murine Pyr12.2 antibody is a neutral valine residue. It is noteworthy that VH9 also contains a K12 residue, and therefore the discrepancy in clearance between H2L7 and H9L8 may not arise from this position. Regarding the light chain, there were no obvious residue differences between VL7 (SEQ ID NO: 23) and VL8 (SEQ ID NO: 24) that were considered to contribute to the increased net positive charge in H2L7 compared to H9L8. Based on sequence alignment, we concluded that the VH domain was likely responsible for the significant difference in clearance between H2L7 and H9L8. Therefore, we hypothesized that mutations in VH2 to remove positively charged residues might reduce the clearance of H2L7 to be more consistent with that observed for H9L8.

[0195] Protein surface analysis of H2L7 and H9L8 revealed a distinct positively charged region in H2L7 that is missing or significantly reduced in H9L8. R83 in the VH domain of H2L7 contributes to a large positively charged patch absent in H9L8 because the corresponding residue at that position (threonine, T83) is a neutral amino acid. Similarly, R71 in the VH domain of H2L7 contributes to a positively charged patch, whereas the corresponding residue in H9L8 is an alanine (A71), which does not contribute to the positively charged protein surface patch. Position 12 in the VH domain of H2L7 was also mutated from K12, present in both VH2 and VH9, back to the neutral V12 present in the murine ancestral Pyr12.2. This may also contribute to a reduction in the overall positively charged protein surface.

[0196] In view of the above analysis, it was hypothesized that variants of H2L7 with the following mutations in the heavy chain variable domain (VH) would have improved PK: H2L7-R71A, H2L7-R83T, H2L7-K12V / R71A, H2L7-K12V / R83T, H2L7-R71A / R83T, and H2L7-K12V / R71A / R83T. The VH sequences are shown in Table 12. The amino acid sequences of the VL and constant domains of the mutant antibodies were identical to the sequences of the corresponding domains in H2L7 shown in Table 6. Among these, H2L7-R71A, H2L7-R83T, H2L7-K12V / R71A, H2L7-K12V / R83T, H2L7-R71A / R83T, and H2L7-K12V / R71A / R83T were produced.

[0197] [Table 14]

[0198] Evaluation of the pharmacokinetic profiles of H2L7 and variants in mice: The plasma PK profiles of H2L7 and five H2L7 variants were evaluated after a single intravenous bolus injection in C57BL / 6 mice. The plasma concentration versus time profiles are shown in Figure 21, and the calculated PK parameters are shown in Table 13. All variants of H2L7 exhibited improved plasma PK profiles in mice, with longer half-lives, higher total exposures (AUC), and lower clearances (CL) than the parent H2L7 antibody.

[0199] [Table 15]

[0200] Example 11 Affinity, selectivity, and specificity of H2L7 mutant antibodies This example describes the characterization of affinity, selectivity, and specificity by inhibition ELISA and SPR of H2L7 mutant antibodies generated and produced as described in Example 10.

[0201] Materials and Methods Aβ monomer species and Aβ protofibrils: The Aβ monomer species described in Example 6 were used to characterize binding to monomeric Aβ species. AβpE3-42 and Aβ1-42 protofibrils were prepared as described in Example 6 and used to characterize binding to aggregated Aβ species.

[0202] Specificity assessment by inhibition ELISA and IC 50 Determination of specificity: Specificity for AβpE3-28 compared to N-terminally intact Aβ (Aβ1-28) and different N-truncated forms of Aβ (Aβ2-28, Aβ3-28, Aβ4-28, Aβ5-28, and AβpE11-28 monomer) was assessed by inhibition ELISA as described for H2L7 and H9L8 in Example 6.

[0203] Selectivity evaluation by inhibition ELISA and IC 50 Determination of: Antibody binding to AβpE3-40 and AβpE3-42 protofibrils and selectivity for Aβ1-40 monomers and Aβ1-42 protofibrils was assessed by inhibition ELISA as described in Example 6 for H2L7 and H9L8.

[0204] Affinity evaluation by surface plasmon resonance and K D Determination: Binding interactions between antigen and antibody were assessed by SPR using a Biacore 8K instrument (Cytiva) according to standard procedures. Binding of the H2L7 mutant antibody to AβpE3-40 monomers and AβpE3-42 protofibrils was assessed as described for H2L7 and H9L8 in Example 6, except that 10 μg / ml of analyte antibody was immobilized on the chip for measurement of binding to AβpE3-40 monomers.

[0205] result Specificity assessment by inhibition ELISA and IC 50Determination of IC The specificity of H2L7, H2L7-K12V / R71A, and H2L7-K12V / R83T for AβpE3-28 compared to N-terminally intact Aβ (Aβ1-28) and different N-truncated forms of Aβ (Aβ2-28, Aβ3-28, Aβ4-28, Aβ5-28, and AβpE11-28 monomer) was assessed by inhibition ELISA. H2L7 and both H2L7 mutants showed the highest binding to soluble AβpE3-28 monomer and some cross-reactivity to Aβ3-28. Calculated IC 50 The values ​​are listed in Table 14.

[0206] [Table 16]

[0207] Selectivity evaluation by inhibition ELISA and IC 50 Determination of IC The binding of H2L7 and five different H2L7 mutants to AβpE3-40 monomers and AβpE3-42 protofibrils, as well as their selectivity for AβpE3-40 monomers and AβpE3-42 protofibrils, was assessed using an inhibition ELISA. H2L7 and its mutants demonstrated binding to dissolved AβpE3-40 monomers and AβpE3-42 protofibrils. None of the antibodies bound to dissolved AβpE3-40 monomers at concentrations up to 500 nM, indicating a corresponding IC . 50 None of the antibodies bound to dissolved Aβ1-42 protofibrils at concentrations up to 132 nM, suggesting that the corresponding IC 50 This suggests that the calculated IC 50 The values ​​are listed in Table 15.

[0208] [Table 17]

[0209] Affinity evaluation by surface plasmon resonance and K DDetermination: The binding of H2L7 and H2L7 mutants to AβpE3-40 monomers and AβpE3-42 protofibrils was assessed by SPR, and their K D value was determined.

[0210] All H2L7 antibody variants showed binding to AβpE3-40 monomers and AβpE3-42 protofibrils. a , k d and (apparent) K D The values ​​are shown below in Tables 16 and 17. Representative sensorgrams are shown in Figures 22 and 23.

[0211] [Table 18]

[0212] [Table 19]

[0213] Example 12 Binding of H2L7 mutant antibodies to targets in brains from human Alzheimer's disease patients and non-demented controls This example describes the target binding of H2L7 and the H2L7 variants generated in Example 10, tested by immunoprecipitation on human brain extracts from AD patients and NDE controls and by immunohistochemistry on human brain sections.

[0214] Materials and Methods Target Binding in Human Alzheimer's Disease Brain Extracts by Immunoprecipitation: Antibody binding to targets in human AD brain was analyzed by immunoprecipitation as described for H2L7 and H9L8 in Example 7.

[0215] Target Binding in Human Alzheimer's Disease Brains by Immunohistochemistry: Immunohistochemistry (IHC) analysis was performed on brain tissue from AD as described for H2L7 and H9L8 in Example 7.

[0216] result Target binding in human Alzheimer's disease brain extracts by immunoprecipitation: Antibody H2L7 and mutants H2L7-K12V / R71A, H2L7-R71A, H2L7-K12V / R83T, H2L7-R71A / R83T, and H2L7-K12V / R71A / R83T were tested for their ability to bind to dissolved AβpE3 in human brain extracts from AD patients. Immunoprecipitation (IP) of TBS brain extracts from AD patients showed concentration-dependent IP of AβpE3-x by all antibodies tested (Figure 24).

[0217] Target binding in human Alzheimer's disease brains by immunohistochemistry: Immunohistochemical staining of brain sections from AD individuals (confirmed to have Aβ pathology by IHC staining with 6E10 / 4G8, not shown) with H2L7 and mutants H2L7-K12V / R71A and H2L7-K12V / R83T resulted in specific binding to nuclear and diffuse plaques in AD brains, with identical staining patterns. No binding was observed in NDE control brains (data not shown). Representative images from immunostaining with H2L7, H2L7-K12V / R71A, and H2L7-K12V / R83T on adjacent sections of fresh-frozen AD brains are shown in Figure 25.

[0218] Example 13 Characterization of the functional effects of H2L7 mutant antibodies This example describes the functional effects of H2L7 mutant antibodies generated as described in Example 10. The ability of the mutant antibodies to inhibit AβpE3 aggregation and clear amyloid plaques in AD brain slices ex vivo was assessed.

[0219] Materials and Methods Inhibition of AβpE3-42 aggregation: The effect of mutant antibodies on the aggregation of AβpE3-42 monomers was assessed in an aggregation assay as described for H2L7 and H9L8 in Example 8.

[0220] Ex vivo phagocytosis in AD brain: An ex vivo phagocytosis assay was used to determine whether the mutant antibodies could induce plaque clearance by macrophages, as described for H2L7 and H9L8 in Example 8.

[0221] result Inhibition of AβpE3-42 aggregation: The ability of the H2L7 mutant antibodies to inhibit AβpE3-42 aggregation was evaluated. All antibodies tested inhibited AβpE3-42 fibril formation in a concentration-dependent manner, as demonstrated by a decrease in the maximum ThT fluorescence signal (Fmax) in the presence of the antibody (Figure 26).

[0222] Ex vivo phagocytosis in AD brain: The ability of the H2L7 mutant antibodies H2L7-K12V / R71A and H2L7-K12V / R83T to induce macrophage clearance of Aβ plaques in AD brains was evaluated. Compared to negative control samples preincubated with no antibody or an isotype control IgG1 antibody, Aβ plaques were significantly reduced after preincubation with H2L7, H2L7-K12V / R71A, and H2L7-K12V / R83T, indicating that the tested antibodies induced plaque clearance by macrophages (Figure 27).

[0223] Example 14 Immunogenicity of H2L7 mutant antibodies This example describes an evaluation of the potential immunogenicity of H2L7 mutant antibodies generated as described in Example 10. The ability of the mutant antibodies to induce CD4+ T cell responses was assessed using an Episcreen™ time course assay.

[0224] Materials and Methods Peripheral blood mononuclear cells (PBMC) isolation: PBMCs were isolated from buffy coats (from blood drawn within 24 hours) of healthy community donors obtained with consent from a commercial supplier. Cells were separated by density centrifugation using lymphocyte separation medium (StemCell Technologies Inc, London, UK), and CD8+ T cells were depleted using CD8+ RosetteSep™ (StemCell Technologies Inc). Donors were characterized by HLA-DR and HLA-DQ haplotype identification to four-digit resolution by Single-Source HLA typing (VHBio, Gateshead, UK). T cell responses to the neoantigen KLH (Invitrogen, Paisley, UK) were also measured. PBMCs were then frozen and stored in nitrogen vapor until needed.

[0225] Sample preparation: Endotoxin levels in antibody samples were measured using an LAL chromogenic kinetic assay kit (Charles River, Margate, UK) according to the manufacturer's instructions and were found to be within acceptable limits for the assay (less than 3 EU / mg).

[0226] Antibody samples were diluted to 0.6 μM in AIM-V® culture medium (Invitrogen) before use (final assay concentration 0.3 μM). KLH was used as a reproducibility control and stored at −20° C. as a 10 mg / ml stock solution in water. For testing, an aliquot of KLH was thawed immediately prior to use and diluted to 200 μg / ml in AIM-V® (final concentration 100 μg / ml). An additional high immunogenicity control, CEFT (a pool of 13 peptides obtained from Pepscan Ltd, Lelystad, The Netherlands), was used as a high-response control and was stored at −20° C. as a 1.538 mg / ml stock solution and diluted to 2 μg / ml in AIM-V® (final concentration 1 μg / ml) before use. Herceptin® (Bionical Ltd, Willington, UK) was used as a negative clinical control and was stored at −80° C. as a 20 mg / ml stock solution (final assay concentration 50 μg / ml).

[0227] Time course proliferation assay: A cohort of 50 donors was selected for the assay. PBMCs from each donor were thawed, counted, and viability assessed using acridine orange (AO) and 4',6-diamidino-2-phenylindole (DAPI) (Chemometec Ltd, Allerod, Denmark) dye exclusion. Cells were revived in room temperature AIM-V® culture medium, washed, and plated at 4-6 x 10 cells in AIM-V® for use as expanded cell stocks. 6The cells were resuspended at 0.3 μM / ml in PBMCs. For each donor, bulk cultures were established by adding 1 ml of expanded cell stock to the appropriate well of a 24-well plate. One ml of each sample was added to PBMCs to achieve a final sample concentration of 0.3 μM. For each donor, a reproducibility control well (cells incubated with 100 μg / ml KLH), an additional high immunogenicity control (cells incubated with 1 μg / ml CEFT peptide pool), a low immunogenicity control (cells incubated with 50 μg / ml Herceptin®), and a well containing culture medium alone were also included. Cultures were incubated at 37°C with 5% CO2 for a total of 8 days. On days 5, 6, 7, and 8, the cells in each well were gently resuspended by mixing 5x using an electronic pipette, and 3 x 100 μl aliquots were transferred to each well of a round-bottom 96-well plate. Cultures were pulsed with 0.75 μCi [H]-thymidine (Perkin Elmer, Beaconsfield, UK) in 100 μl AIM-V® culture medium and incubated for a further 18 hours before harvesting onto filter mats (Perkin Elmer) using a TomTec Mach III cell harvester. CPM for each well was determined by Meltilex™ (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux liquid scintillation counter (Perkin Elmer) with Paralux low background counting.

[0228] Assessment of cell viability: On day 7, bulk cultures (already established for proliferation assays) were gently resuspended by mixing 5x with an electronic pipette, and 50 μl was removed from each well and mixed with 2.5 μl acridine orange (AO) and 4',6-diamidino-2-phenylindole (DAPI) (Chemometec Ltd) dye exclusion. Cell viability was then assessed using a NucleoCounter® NC-250™ automated cell analyzer (Chemometec Ltd).

[0229] Data Analysis: For proliferation assays, an empirical threshold of stimulation index (SI) of 1.9 or greater (SI≧1.90) was previously established, whereby samples inducing a response above this threshold were considered positive. For proliferation analysis, donors who were positive at at least one time point during the time course assay were considered positive donors and added to the "% response" parameter.

[0230] result The ability of the H2L7 mutant antibodies H2L7-K12V / R71A and H2L7-K12V / R83T to induce CD4+ T cell responses was evaluated. Reference antibodies A and B were used for comparison. Reference antibody A comprises the heavy chain amino acid sequence SEQ ID NO: 33 and the light chain amino acid sequence SEQ ID NO: 34. Reference antibody B comprises the heavy chain amino acid sequence SEQ ID NO: 35 and the light chain amino acid sequence SEQ ID NO: 36.

[0231] EpiScreen™ analysis of response frequency and magnitude showed that H2L7-K12V / R71A and H2L7-K12V / R83T induced responses slightly higher than those of the low-immunogenicity control Herceptin® and were therefore considered to pose a relatively low risk of immunogenicity. Both reference antibodies A and B induced significantly higher proliferative responses compared to Herceptin® and were therefore considered to pose a high risk of immunogenicity in clinical settings.

[0232] [Table 20]

[0233] Itemized List of Embodiments 1. An antibody having affinity for AβpE3, wherein the six complementarity determining regions of the heavy and light chain variable domains have the following amino acid sequences: VH-CDR1: GX1TX2N (SEQ ID NO: 1) (In the formula, X1 is selected from Y and F; and X2 is selected from L and M; VH-CDR2: LINPYNGX3TTYNX4KFX5G (SEQ ID NO: 2) (In the formula, X3 is selected from I and V X4 is selected from P and Q; and X5 is selected from M and K; VH-CDR3: EGNWEGVY (SEQ ID NO: 3) VL-CDR1: X6SSQSLLDSNGKTYLH (SEQ ID NO: 4) (In the formula, X6 is selected from K and R; VL-CDR2: LVSX7LDS (SEQ ID NO: 5) (In the formula, X7 is selected from I and K; VL-CDR3: VQGTHFPFT (SEQ ID NO: 6) An antibody or antigen-binding fragment thereof comprising:

[0234] 2. The VH-CDR1, VH-CDR2 and VL-CDR2 regions have the following amino acid sequences: VH-CDR1: GFTMN (SEQ ID NO: 7) VH-CDR2: LINPYNGVTTYNQKFKG (SEQ ID NO: 8) VL-CDR2: LVSILDS (SEQ ID NO: 9) 2. The antibody or antigen-binding fragment thereof according to item 1,

[0235] 3. A heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is i) the group consisting of SEQ ID NOs: 15 to 22; and ii) an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 15 to 22, provided that the three VH-CDR regions consist of SEQ ID NOs: 7, 8, and 3; The antibody or antigen-binding fragment thereof according to any of the preceding items, comprising an amino acid sequence selected from:

[0236] 4. The antibody or antigen-binding fragment thereof according to item 3, wherein the VH amino acid sequence in i) is selected from the group consisting of SEQ ID NOs: 15 to 21.

[0237] 5. The antibody or antigen-binding fragment thereof according to item 4, wherein the VH amino acid sequence in i) is selected from the group consisting of SEQ ID NOs: 15 to 16 and 18 to 21.

[0238] 6. The antibody or antigen-binding fragment thereof according to item 4, wherein the VH amino acid sequence in i) is selected from the group consisting of SEQ ID NOs: 15 to 20.

[0239] 7. The antibody or antigen-binding fragment thereof according to any one of items 5 to 6, wherein the VH amino acid sequence in i) is selected from the group consisting of SEQ ID NOs: 15 to 16 and 18 to 20.

[0240] 8. The antibody or antigen-binding fragment thereof according to item 7, wherein the VH amino acid sequence in i) is selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 18.

[0241] 9. The antibody or antigen-binding fragment thereof according to item 8, wherein the VH amino acid sequence in i) is SEQ ID NO: 18.

[0242] 10. The amino acid sequence of VL-CDR1 is RSSQSLLDSNGKTYLH (SEQ ID NO: 10) The antibody or antigen-binding fragment thereof according to any of the preceding items,

[0243] 11. A heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain is i) the group consisting of SEQ ID NOs: 23 to 24; and ii) an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 23 to 24, provided that the three VL-CDR regions consist of SEQ ID NOs: 10, 9, and 6; 11. The antibody or antigen-binding fragment thereof according to item 10, comprising an amino acid sequence selected from the group consisting of:

[0244] 12. The antibody or antigen-binding fragment thereof according to item 11, wherein the VL amino acid sequence in i) is SEQ ID NO: 23.

[0245] 13. The antibody or antigen-binding fragment thereof according to any of the preceding items, wherein the heavy chain variable domain is as defined in any one of items 3 to 9, and the light chain variable domain is as defined in any one of items 11 to 12.

[0246] 14. The heavy chain variable domain and the light chain variable domain have the following VH / VL combination: a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; f) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; g) a heavy chain variable domain comprising SEQ ID NO: 21 and a light chain variable domain comprising SEQ ID NO: 24; h) a heavy chain variable domain comprising SEQ ID NO: 22 and a light chain variable domain comprising SEQ ID NO: 23 14. The antibody or antigen-binding fragment according to item 13, wherein the antibody or antigen-binding fragment is represented by

[0247] 15. The heavy chain variable domain and the light chain variable domain have the following VH / VL combination: a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; f) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; g) a heavy chain variable domain comprising SEQ ID NO: 21 and a light chain variable domain comprising SEQ ID NO: 24 15. The antibody or antigen-binding fragment thereof according to item 14, wherein the antibody or antigen-binding fragment thereof is represented by

[0248] 16. The heavy chain variable domain and the light chain variable domain have the following VH / VL combination: a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; g) a heavy chain variable domain comprising SEQ ID NO: 21 and a light chain variable domain comprising SEQ ID NO: 24 16. The antibody or antigen-binding fragment according to item 15, wherein the antibody or antigen-binding fragment is represented by

[0249] 17. The heavy chain variable domain and the light chain variable domain have the following VH / VL combination: a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; f) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23 16. The antibody or antigen-binding fragment according to item 15, wherein the antibody or antigen-binding fragment is represented by

[0250] 18. The heavy chain variable domain and the light chain variable domain have the following VH / VL combination: a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23 18. The antibody or antigen-binding fragment according to any one of items 16 to 17, wherein the antibody or antigen-binding fragment is represented by

[0251] 19. The heavy chain variable domain and the light chain variable domain have the following VH / VL combination: a) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23 19. The antibody or antigen-binding fragment according to item 18, wherein the antibody or antigen-binding fragment is represented by

[0252] 20. The heavy chain variable domain and the light chain variable domain have the following VH / VL combination: a) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23 20. The antibody or antigen-binding fragment according to item 19, wherein the antibody or antigen-binding fragment is represented by

[0253] 21. The amino acid sequence of VL-CDR1 is KSSQSLLDSNGKTYLH (SEQ ID NO: 11), 10. The antibody or antigen-binding fragment according to any one of items 1 to 9.

[0254] 22. A heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is: i) SEQ ID NO: 25; and ii) an amino acid sequence having at least 80% identity with SEQ ID NO: 25, provided that the three VH-CDR regions consist of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 3; 22. The antibody or antigen-binding fragment thereof according to item 21, comprising an amino acid sequence selected from the group consisting of:

[0255] 23. A heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain is: i) SEQ ID NO: 26; and ii) an amino acid sequence having at least 80% identity with SEQ ID NO: 26, provided that the three VL-CDR regions consist of SEQ ID NO: 11, SEQ ID NO: 9 and SEQ ID NO: 6; 23. The antibody or antigen-binding fragment thereof according to any one of items 21 to 22, comprising an amino acid sequence selected from the group consisting of:

[0256] 24. The antibody or antigen-binding fragment thereof according to any one of items 22 to 23, wherein the heavy chain variable domain is as defined in item 22 and the light chain variable domain is as defined in item 23.

[0257] 25. The VH-CDR1, VH-CDR2, VL-CDR1 and VL-CDR2 regions have the following amino acid sequences: VH-CDR1: GYTLN (SEQ ID NO: 12); VH-CDR2: LINPYNGITTYNPKFMG (SEQ ID NO: 13); VL-CDR1: KSSQSLLDSNGKTYLH (SEQ ID NO: 11) VL-CDR2: LVSKLDS (SEQ ID NO: 14) 2. The antibody or antigen-binding fragment thereof according to item 1,

[0258] 26. A heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is: i) SEQ ID NO: 27; and ii) an amino acid sequence having at least 80% identity with SEQ ID NO: 27, provided that the three VH-CDR regions consist of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 3; 26. The antibody or antigen-binding fragment thereof according to item 25, comprising an amino acid sequence selected from the group consisting of:

[0259] 27. A heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain is: i) SEQ ID NO: 28; and ii) an amino acid sequence having at least 80% identity with SEQ ID NO: 28, provided that the three VL-CDR regions consist of SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 6; 27. The antibody or antigen-binding fragment thereof according to any one of items 25 to 26, comprising an amino acid sequence selected from the group consisting of:

[0260] 28. The antibody or antigen-binding fragment thereof according to any one of items 26 to 27, wherein the heavy chain variable domain is as defined in item 26 and the light chain variable domain is as defined in item 27.

[0261] 29. The antibody or antigen-binding fragment thereof according to any one of the preceding items, wherein the AβpE3 is in a form selected from the group consisting of monomers, protofibrils, fibrils, and plaques.

[0262] 30. The antibody or antigen-binding fragment thereof according to any of the preceding items, which has higher binding affinity for AβpE3 monomers than for Aβ1-X monomers.

[0263] 31. The antibody or antigen-binding fragment thereof according to item 30, which has a binding affinity for AβpE3 monomer that is at least 2-fold higher, such as at least 10-fold higher, such as at least 100-fold higher, such as at least 1000-fold higher, such as at least 3000-fold higher than for Aβ1-X monomer.

[0264] 32. The antibody or antigen-binding fragment thereof according to any of the preceding items, which has higher binding affinity for AβpE3-containing protofibrils than for AβpE3 monomers.

[0265] 33. The antibody or antigen-binding fragment thereof according to item 24, which has a binding affinity for AβpE3-containing protofibrils that is at least 2-fold higher, such as at least 10-fold higher, such as at least 40-fold higher, such as at least 100-fold higher, such as at least 200-fold higher than for AβpE3 monomers.

[0266] 34. A K of 1 nM or less, e.g., 1 to 200 pM, e.g., 10 to 100 pM, as determined by surface plasmon resonance. D The antibody or antigen-binding fragment thereof according to any of the preceding items, having a binding affinity for protofibrils containing AβpE3 corresponding to a value.

[0267] 35. A K of 100 nM or less, e.g., 0.1 to 50 nM, e.g., 0.5 to 10 nM, as determined by surface plasmon resonance. D The antibody or antigen-binding fragment thereof according to any of the preceding items, having a binding affinity for AβpE3 monomer corresponding to a value.

[0268] 36. An antigen-binding fragment according to any of the preceding items, which is selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain Fv fragments, (scFv)2 and domain antibodies.

[0269] 37. An antibody or antigen-binding fragment thereof according to any of the preceding items, which is an IgG class antibody.

[0270] 38. The antibody or antigen-binding fragment thereof according to item 37, wherein the IgG class is selected from the group consisting of IgG1 and IgG4.

[0271] 39. The antibody or antigen-binding fragment thereof according to any of the preceding items, which is monoclonal.

[0272] 40. The antibody or antigen-binding fragment thereof according to any of the preceding items, selected from the group consisting of human antibodies and fragments thereof; humanized antibodies and fragments thereof; and antibodies and fragments thereof that have been mutated to reduce their antigenicity in humans.

[0273] 41. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any of the preceding items and a pharmaceutically acceptable carrier or excipient.

[0274] 42. The antibody or antigen-binding fragment thereof according to any one of items 1 to 40 or the composition according to item 41 for use in treatment, such as for use in therapeutic treatment or for use in prophylactic treatment.

[0275] 43. The antibody or antigen-binding fragment thereof according to any one of items 1 to 40 or the composition according to item 41 for use in in vivo diagnosis or in vivo prognosis.

[0276] 44. The antibody or antigen-binding fragment thereof, or composition for use according to any one of items 42 to 43, wherein the treatment, prevention, in vivo diagnosis, or in vivo prognosis is for a neurodegenerative disorder associated with amyloid beta peptide aggregation, for example, a disorder selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), dementia with Lewy bodies, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataracts due to Aβ deposition, traumatic brain injury associated with Aβ accumulation, adult-onset diabetes, senile cardiac amyloidosis, and macular degeneration.

[0277] 45. The antibody or antigen-binding fragment thereof for use according to item 44, wherein the neurodegenerative disorder is Alzheimer's disease.

[0278] 46. ​​A method for therapeutic or prophylactic treatment of a mammal having or at risk of developing a neurodegenerative disorder, comprising administering to said mammal a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 40 or the composition according to item 41.

[0279] 47. The method according to item 46, wherein the neurodegenerative disorder is a disorder associated with amyloid beta peptide aggregation, for example, a disorder selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), dementia with Lewy bodies, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebrovascular disease with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataracts due to Aβ deposition, traumatic brain injury associated with Aβ accumulation, adult-onset diabetes, senile cardiac amyloidosis, and macular degeneration.

[0280] 48. The method according to item 47, wherein the neurodegenerative disorder is Alzheimer's disease.

[0281] 49. A method for detecting AβpE3 peptide in vitro, comprising providing a sample suspected of containing Aβ peptide, contacting the sample with an antibody or antigen-binding fragment thereof according to any one of items 1 to 40, and detecting binding of the protein, thereby indicating the presence of AβpE3 peptide in the sample.

[0282] 50. A method for determining the amount of AβpE3 peptide present in a subject, comprising: a) contacting a subject or a sample isolated from a subject with the antibody or antigen-binding fragment thereof according to any one of items 1 to 40 or the composition according to item 41; and b) obtaining a value corresponding to the amount of bound antibody or antigen-binding fragment thereof or composition in said subject or said sample; A method comprising:

[0283] 51. The method according to item 50, further comprising the step of comparing the value with a reference.

Claims

1. An antibody having affinity for AβpE3, wherein the six complementarity determining regions of the heavy and light chain variable domains have the following amino acid sequences: VH-CDR1:GX １ TX ２ N (SEQ ID NO: 1) (In the formula, X １ is selected from Y and F; and X ２ is selected from L and M; VH-CDR2:LINPYNGX ３ TTYNX ４ KFX ５ G (SEQ ID NO: 2) (In the formula, X ３ is selected from I and V X ４ is selected from P and Q; and X ５ is selected from M and K; VH-CDR3: EGNWEGVY (SEQ ID NO: 3) VL-CDR1:X ６ SSQSLLDSNGKTYLH (SEQ ID NO: 4) (In the formula, X ６ is selected from K and R; VL-CDR2: LVSX ７ LDS (SEQ ID NO: 5) (In the formula, X ７ is selected from I and K; VL-CDR3: VQGTHFPFT (SEQ ID NO: 6) An antibody consisting of, or an antigen-binding fragment thereof.

2. The VH-CDR1, VH-CDR2 and VL-CDR2 regions have the following amino acid sequences: VH-CDR1: GFTMN (SEQ ID NO: 7) VH-CDR2: LINPYNGVTTYNQKFKG (SEQ ID NO: 8) VL-CDR2: LVSILDS (SEQ ID NO: 9) The antibody or antigen-binding fragment thereof according to claim 1, comprising:

3. a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: i) the group consisting of SEQ ID NOs: 15-22; and ii) an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 15 to 22, provided that the three VH-CDR regions consist of SEQ ID NOs: 7, 8 and 3; The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising an amino acid sequence selected from:

4. the amino acid sequence of VL-CDR1 is RSSQSLLDSNGKTYLH (SEQ ID NO: 10) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3,

5. a heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain comprises: i) the group consisting of SEQ ID NOs: 23-24; and ii) an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 23-24, provided that the three VL-CDR regions consist of SEQ ID NOs: 10, 9, and 6; The antibody or antigen-binding fragment thereof of claim 4, comprising an amino acid sequence selected from:

6. The heavy chain variable domain and the light chain variable domain have the following VH / VL combinations: a) a heavy chain variable domain comprising SEQ ID NO: 15 and a light chain variable domain comprising SEQ ID NO: 23; b) a heavy chain variable domain comprising SEQ ID NO: 16 and a light chain variable domain comprising SEQ ID NO: 23; c) a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 23; d) a heavy chain variable domain comprising SEQ ID NO: 18 and a light chain variable domain comprising SEQ ID NO: 23; e) a heavy chain variable domain comprising SEQ ID NO: 19 and a light chain variable domain comprising SEQ ID NO: 23; f) a heavy chain variable domain comprising SEQ ID NO: 20 and a light chain variable domain comprising SEQ ID NO: 23; g) a heavy chain variable domain comprising SEQ ID NO: 21 and a light chain variable domain comprising SEQ ID NO: 24; h) a heavy chain variable domain comprising SEQ ID NO: 22 and a light chain variable domain comprising SEQ ID NO: 23 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is represented by:

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the AβpE3 is in a form selected from the group consisting of monomers, protofibrils, fibrils, and plaques.

8. A K of 1 nM or less, e.g., 1-200 pM, e.g., 10-100 pM, as determined by surface plasmon resonance. Ｄ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, having a binding affinity to protofibrils containing AβpE3 equivalent to a value

9. a K of 100 nM or less, e.g., 0.1 to 50 nM, e.g., 0.5 to 10 nM, as determined by surface plasmon resonance Ｄ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, having a binding affinity for AβpE3 monomer corresponding to a value of

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier or excipient.

11. 11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9 or the composition of claim 10 for use in treatment, such as for use in therapeutic treatment or for use in prophylactic treatment.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the composition according to claim 10 for use in in vivo diagnosis or in in vivo prognosis.

13. 13. The antibody or antigen-binding fragment thereof, or composition for use according to claim 11 or 12, wherein the treatment, prevention, in vivo diagnosis or in vivo prognosis is for a neurodegenerative disorder associated with amyloid beta peptide aggregation, such as a disorder selected from the group consisting of Alzheimer's disease (AD) (including familial AD and sporadic AD), mild cognitive impairment (MCI), dementia with Lewy bodies, neurodegeneration in Down's syndrome, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis (Dutch type), progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, cerebral amyloid angiopathy, Parkinson's disease, amyotrophic lateral sclerosis, cataracts due to Aβ deposition, traumatic brain injury associated with Aβ accumulation, adult-onset diabetes mellitus, senile cardiac amyloidosis, and macular degeneration.

14. The antibody or antigen-binding fragment thereof for use according to claim 13, wherein the neurodegenerative disorder is Alzheimer's disease.

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