Anti-amyloid beta fibril / oligomer antibodies and uses thereof

Abstract

The present disclosure provides anti-amyloid beta (Aβ) antibodies and antibody fragments that preferentially bind to soluble amyloid Aβ fibrils / oligomers and trigger ADPCs in microglia; anti-amyloid beta (Aβ) antibodies and antibody fragments that reduce soluble amyloid Aβ fibril / oligomer levels and insoluble amyloid Aβ plaques in brain tissue; and uses of anti-Aβ fibril / oligomer antibodies and antibody fragments in the treatment, prevention, diagnosis, screening and monitoring of conditions associated with Aβ protein aggregation, in particular Alzheimer's disease (AD).

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 440,345, filed January 20, 2023, entitled “Anti-amyloid β-oligomer antibodies and uses thereof,” the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list that has been electronically submitted in XML file format, which is hereby incorporated in its entirety by reference. The XML copy created on January 17, 2024, is named 1121-103PCT_SL.xml and has a size of 187,258 bytes. Technical Field

[0005] This disclosure relates to antibodies and antibody fragments that preferentially bind to soluble amyloid β (Aβ) fibrils / oligomers, and the use of anti-Aβ fibrils / oligomer antibodies and antibody fragments in the treatment, prevention, diagnosis, screening, and monitoring of symptoms associated with Aβ protein aggregation, particularly in Alzheimer's disease (AD). Background Technology

[0006] Neurodegenerative conditions associated with the aggregation and deposition of amyloid-β (Aβ) protein include Alzheimer's disease (AD), a common type of neurodegenerative disease. AD patients eventually lose their ability to perform daily activities due to memory loss, mood / behavioral changes, and other disabilities. In the United States, more than 6 million Americans have AD, and this number is projected to reach 14 million by 2060. Globally, more than 55 million people suffer from dementia, with 60-70% of cases potentially related to AD. In 2019, the global economic burden of AD and related dementia was estimated at $2.8 trillion. This figure is projected to increase to $16.9 trillion by 2050. Seven FDA-approved drugs exist for AD, but none of these drugs have shown any benefit in altering the disease.

[0007] AD is classified into familial AD (fAD) and late-onset AD (LOAD). Mutations in APP and PSEN1 have been reported to be the cause of fAD. Both genes are responsible for the production of Aβ peptides, which are released from amyloid-β precursor protein (APP) through proteolytic cleavage involving presenile protein 1 encoded by the PSEN1 gene. The cause of LOAD is unclear. Amyloid plaques and tau tangles are the most prominent pathological features of the AD brain. Amyloid plaques are aggregates of Aβ deposits and usually form before tau tangles. The onset of LOAD is highly correlated with the presence of amyloid deposits, and the severity or stage of the disease is correlated with the level of tau tangles. Therefore, major efforts in the development of AD treatment have focused on targeting Aβ deposits and tau tangles. Aβ may be a better therapeutic target for AD than tau for the following reasons: (1) Aβ exists in the extracellular space and may be more easily accessed by biologics than tau tangles, and (2) Aβ pathology is upstream of tau pathology.

[0008] Aβ peptides exist in various forms, including monomeric, aggregated (including soluble fibrils / oligomers), and insoluble plaques. Soluble fibrils / oligomers are considered neurotoxic, while insoluble plaques resulting from the deposition of aggregated Aβ fibers are considered inert. Aβ has been a major therapeutic target for AD drug discovery over the past 20 years. Both monomeric and aggregated forms of Aβ have been targeted. To date, monoclonal antibodies targeting Aβ monomers have not cleared amyloid plaques or provided cognitive benefits. A total of five (5) mAbs targeting extracellular aggregated Aβ (aducanumab, crenezumab, lecanemab, donanemab, and gantenerumab) have been tested in phase 2 or 3 clinical trials. With the exception of one antibody (crirnizumab), all of these antibodies effectively cleared amyloid plaques in human patients in a dose-dependent manner (crirnizumab is an IgG4 isotype and may not have strong effector function). Aducanumab and lencanemumab have been reported to slow cognitive decline in a phase 3 trial. Donemumab has been reported to slow cognitive decline in a phase 2 trial. These data confirm that monoclonal antibodies can reach their targets in the brain when used at high doses, and that aggregated Aβ may be a plausible target for AD. Lencanemumab, which preferentially binds to Aβ fibrils / oligomers, had a lower incidence of edema than aducanumab and gantenerumab, which preferentially bind to Aβ plaques. Summary of the Invention

[0009] This disclosure provides anti-Aβ fibril / oligomer antibodies and antibody fragments that preferentially bind to Aβ fibrils / oligomers, amino acid sequences of variable regions of the anti-Aβ fibril / oligomer antibodies and antibody fragments, nucleotide sequences encoding the variable regions of the anti-Aβ fibril / oligomer antibodies and antibody fragments, and methods for identifying, preparing, and using the anti-Aβ fibril / oligomer antibodies and antibody fragments. The anti-Aβ fibril / oligomer antibodies and antibody fragments provided herein have fully human variable regions having fully human heavy chain variable region (VH) sequences and fully human light chain variable region (VL) sequences. This disclosure further provides fully human anti-Aβ fibril / oligomer antibodies and antibody fragments that preferentially bind to Aβ fibrils / oligomers, wherein the fully human anti-Aβ fibril / oligomer antibodies and antibody fragments have fully human heavy chain variable region and fully human light chain variable region sequences.

[0010] This disclosure provides anti-Aβ fibrils / oligomery antibodies and antibody fragments thereof that preferentially bind to soluble Aβ fibrils / oligomers, wherein the anti-Aβ fibrils / oligomery antibodies and antibody fragments can trigger antibody-dependent cell-mediated phagocytosis (ADCP) in cells including microglia exposed to the anti-Aβ fibrils / oligomery antibodies and antibody fragments bound to Aβ fibrils / oligomers. Anti-Aβ fibrils / oligomery antibodies and antibody fragments thereof are provided that can penetrate brain tissue after administration to a mammalian individual. Anti-Aβ fibrils / oligomery antibodies and antibody fragments thereof are provided that can reduce soluble Aβ oligomers / fibrils in brain tissue after administration to a mammalian individual. Anti-Aβ fibrils / oligomery antibodies and antibody fragments thereof are provided, which can reduce insoluble Aβ fibrils / plaques in brain tissue, including both condensed and diffuse plaques, after administration to mammalian individuals. The anti-Aβ fibrils / oligomery antibodies and antibody fragments with fully human variable regions provided herein have a lower risk of immunogenicity-triggered adverse reactions or undesirable side effects when administered to mammalian individuals, particularly human individuals.

[0011] This disclosure provides anti-Aβ fibrils / oligomery antibodies and antibody fragments for methods of treating, preventing, diagnosing, screening, and monitoring conditions associated with Aβ protein aggregation (including, but not limited to, Alzheimer's disease (AD)).

[0012] This disclosure provides anti-Aβ fibrils / oligomery antibodies and antibody fragments comprising an amino acid sequence that is at least 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to one of the following: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 49; SEQ ID NO: 51; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:62; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:66; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:82; SEQ ID NO:83; SEQ ID NO:84; SEQ ID SEQ ID NO: 87; SEQ ID NO: 89; SEQ ID NO: 91; SEQ ID NO: 92; SEQ ID NO: 93; SEQ ID NO: 94; SEQ ID NO: 96; SEQ ID NO: 97; SEQ ID NO: 99;SEQ IDNO:101; SEQ ID NO:102; SEQ ID NO:103; SEQ ID NO:104; SEQ ID NO:106; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:112; SEQ ID NO:113; SEQ ID NO:114; SEQ IDNO:116; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:122; SEQ ID NO:123; SEQ ID NO:124; SEQ ID NO:126; SEQ ID NO:127; SEQ ID NO:129; SEQ ID NO:131; SEQ IDNO:132; SEQ ID NO:133; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:142; SEQ ID NO:143; SEQ ID NO:144; SEQ ID NO:146; SEQ IDNO:147; and SEQ ID NO:149.;

[0013] This disclosure provides anti-Aβ fibrils / oligomery antibodies and antibody fragments identified as follows: antibodies and antibody fragments comprising an 06E17A variable region, the variable region comprising a fully human heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO:1, and a fully human light chain variable region (VL) having the amino acid sequence of SEQ ID NO:6; antibodies and antibody fragments comprising a 15M13A variable region, the variable region comprising a fully human VH having the amino acid sequence of SEQ ID NO:11, and a fully human VL having the amino acid sequence of SEQ ID NO:16; antibodies and antibody fragments comprising a 17D08A variable region, the variable region comprising a fully human VH having the amino acid sequence of SEQ ID NO:21, and a fully human VL having the amino acid sequence of SEQ ID NO:1. The following are examples of antibodies and antibody fragments: antibody and antibody fragment comprising a 17H05A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:31, and a fully human VL having the amino acid sequence of SEQ ID NO:36; antibody and antibody fragment comprising a 17P04A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:41, and a fully human VL having the amino acid sequence of SEQ ID NO:46; antibody and antibody fragment comprising an 18F06A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:51, and a fully human VL having the amino acid sequence of SEQ ID NO:56; antibody and antibody fragment comprising an 18P01A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:26. The following are listed: an amino acid sequence of SEQ ID NO:61, and a fully human VL having the amino acid sequence of SEQ ID NO:66; an antibody and an antibody fragment comprising a 20O07A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:71, and a fully human VL having the amino acid sequence of SEQ ID NO:76; an antibody and an antibody fragment comprising a 20O11A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:81, and a fully human VL having the amino acid sequence of SEQ ID NO:86;Antibodies and antibody fragments, the antibodies and antibody fragments comprising a 21F12A variable region, the variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:91, and a fully human VL having the amino acid sequence of SEQ ID NO:96; antibodies and antibody fragments comprising a 21G10A variable region, the variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:101, and a fully human VL having the amino acid sequence of SEQ ID NO:106; antibodies and antibody fragments comprising a 21K12A variable region, the variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:111, and a fully human VL having the amino acid sequence of SEQ ID NO:116; antibodies and antibody fragments comprising a 21P22A variable region, the variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:91, and the fully human VL having the amino acid sequence of SEQ ID NO:96. The following are listed: an amino acid sequence of SEQ ID NO:121, and a fully human VL having the amino acid sequence of SEQ ID NO:126; an antibody and an antibody fragment comprising a 22D04A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:131, and a fully human VL having the amino acid sequence of SEQ ID NO:136; an antibody and an antibody fragment comprising a 22H10A variable region containing a fully human VH having the amino acid sequence of SEQ ID NO:141, and a fully human VL having the amino acid sequence of SEQ ID NO:146; and an anti-Aβ oligomer antibody and an antibody fragment having at least 85% amino acid sequence identity with the above-described VH and / or VL amino acid sequences.

[0014] This disclosure provides the following anti-Aβ fibrils / oligomery antibodies and antibody fragments: (a) an antibody or antibody fragment comprising a heavy chain variable region (VH) having an HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO:42), an HC CDR2 having the amino acid sequence ISSRTYI (SEQ ID NO:43), and an HC CDR3 having the amino acid sequence ARGGYIGSPNAYDI (SEQ ID NO:44), and a light chain variable region (VL) having an LC CDR1 having the amino acid sequence TGAVTSDYY (SEQ ID NO:47), an LC CDR2 having the amino acid sequence SAS, and an LC CDR3 having the amino acid sequence LLYYGGAWV (SEQ ID NO:49); (b) an antibody or antibody fragment comprising a VH having an HC CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO:62), an HC CDR2 having the amino acid sequence IRSKANSYAT (SEQ ID NO:63), and an HC CDR3 having the amino acid sequence GFTFSGSA (SEQ ID NO:63). (c) An antibody or antibody fragment comprising VH, wherein the VH comprises HC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 67), HC CDR2 having the amino acid sequence DNS, and HC CDR3 having the amino acid sequence GTWDSSLSTYV (SEQ ID NO: 69); and (d) an antibody or antibody fragment comprising VH, wherein the VH comprises HC CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO: 72), HC CDR2 having the amino acid sequence IRSKVNSYAT (SEQ ID NO: 73), and HC CDR3 having the amino acid sequence TSHAPIFDAFDI (SEQ ID NO: 74), and VL, wherein the VL comprises LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 77), LC CDR2 having the amino acid sequence DNS, and LC CDR3 having the amino acid sequence GTWDSSLSTYV (SEQ ID NO: 79);(d) An antibody or antibody fragment comprising VH, wherein the VH comprises HC CDR1 having the amino acid sequence GFTFSNAW (SEQ ID NO: 132), HC CDR2 having the amino acid sequence IKSKTDGGTR (SEQ ID NO: 133), and HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 134), and VL comprising LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO: 137), LCCDR2 having the amino acid sequence RNN, and LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 139); and (e) an antibody or antibody fragment comprising VH, wherein the VH comprises HCCDR1 having the amino acid sequence GFSFSNAW (SEQ ID NO: 2), HC CDR2 having the amino acid sequence IKSKTDGGTI (SEQ ID NO: 3), and HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 4). CDR3, and VL, wherein VL comprises LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO:7), LC CDR2 having the amino acid sequence RNN, and LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO:9).

[0015] This disclosure provides anti-Aβ fibrils / oligomery antibodies and antibody fragments having a constant region sequence, said constant region sequence including a fragment crystallizable region (Fc) sequence sufficient to achieve Fc-mediated effector functions such as antibody-dependent phagocytosis (ADCP) . This disclosure provides anti-Aβ fibrils / oligomery antibodies and antibody fragments having an Fc sequence derived from IgG class immunoglobulin molecules. This disclosure provides anti-Aβ fibrils / oligomery antibodies and antibody fragments having a constant region Fc, said constant region Fc may be mouse IgG1 Fc, human IgG1 Fc, mouse IgG2a Fc, and mouse IgG2b Fc.

[0016] This disclosure provides pharmaceutical compositions comprising at least one anti-Aβ fibrils / oligomery antibody or antibody fragment as disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0017] This disclosure provides a method for reducing the amount of soluble Aβ fibrils / oligomers in an individual by administering to the individual a therapeutically effective amount of at least one of the anti-Aβ fibrils / oligomer antibodies or antibody fragments disclosed herein. A method for reducing the amount of soluble Aβ fibrils / oligomers in an individual is provided, wherein administering to the individual a therapeutically effective amount of at least one of the anti-Aβ fibrils / oligomer antibodies or antibody fragments disclosed herein triggers antibody-dependent cell-mediated phagocytosis (ADCP) of a complex comprising the anti-Aβ fibrils / oligomer antibody or antibody fragment bound to the Aβ fibrils / oligomers. ADCP mediated by response effector cells can occur in any tissue, including at least one of blood, lymph, cerebrospinal fluid (CSF), neural tissue, and the brain.

[0018] This disclosure provides a method for reducing the amount of soluble Aβ fibrils / oligomers in the brain of a mammalian individual by administering a therapeutically effective amount of at least one of the anti-Aβ fibrils / oligomer antibodies or antibody fragments disclosed herein.

[0019] This disclosure provides a method for reducing the amount of Aβ plaques in an individual, the method comprising administering to the individual a therapeutically effective amount of the anti-Aβ fibrils / oligomery antibody or antibody fragment disclosed herein. This disclosure provides a method for reducing the level of Aβ plaques in the brain of a mammalian individual by administering to the individual a therapeutically effective amount of at least one of the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein. This disclosure provides a method for reducing the level of concentrated Aβ plaques in the brain of a mammalian individual by administering to the individual a therapeutically effective amount of at least one of the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein. This disclosure provides a method for reducing the level of diffuse Aβ plaques in the brain of a mammalian individual by administering to the individual a therapeutically effective amount of at least one of the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein. Therefore, this disclosure provides a method for reducing plaque burden in an individual, the method comprising administering to the individual a therapeutically effective amount of the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein. A method for reducing the amount of Aβ plaque in an individual is provided, wherein administering a therapeutically effective amount of at least one anti-Aβ fibril / oligomer antibody or antibody fragment disclosed herein to the individual is expected to trigger ADCP of a complex comprising the anti-Aβ fibril / oligomer antibody or antibody fragment that binds to Aβ fibrils / oligomers, resulting in a reduction in the amount of Aβ plaque in the individual.

[0020] This disclosure provides a method for treating at least one symptom associated with Aβ protein aggregation by administering an effective amount of at least one anti-Aβ oligomer antibody or antibody fragment as disclosed herein to an individual in need. A method is provided for treating at least one symptom associated with Aβ protein aggregation by administering an effective amount of at least one anti-Aβ fibril / oligomer antibody or antibody fragment thereof, wherein the at least one symptom associated with Aβ protein aggregation is Alzheimer's disease (AD).

[0021] This disclosure provides a method for treating at least one symptom associated with Aβ protein aggregation, wherein administering to an individual in need at least one anti-Aβ fibril / oligomer antibody or antibody fragment disclosed herein to trigger antibody-dependent cell-mediated phagocytosis (ADCP) of a complex comprising the anti-Aβ fibril / oligomer antibody or antibody fragment bound to the Aβ fibril / oligomer.

[0022] This disclosure provides a method for preventing the onset or further development of at least one symptom associated with Aβ protein aggregation by administering an effective amount of at least one anti-Aβ fibril / oligomer antibody or antibody fragment as disclosed herein to an individual in need. A method for preventing the onset or further development of at least one symptom associated with Aβ protein aggregation is provided, wherein administering the anti-Aβ fibril / oligomer antibody or antibody fragment to an individual in need triggers antibody-dependent cell-mediated phagocytosis (ADCP) of a complex comprising the anti-Aβ fibril / oligomer antibody or antibody fragment bound to Aβ fibrils / oligomers, thereby substantially preventing Aβ protein aggregation in the individual and ultimately reducing plaque burden. A method for treating at least one symptom associated with Aβ protein aggregation, preventing the onset of at least one symptom, or preventing further development of at least one symptom associated with Aβ protein aggregation is provided by administering an effective amount of at least one anti-Aβ fibril / oligomer antibody or antibody fragment, wherein the at least one symptom associated with Aβ protein aggregation is Alzheimer's disease (AD).

[0023] This disclosure provides a method for diagnosing at least one symptom associated with Aβ protein aggregation in an individual by detecting Aβ fibrils / oligomers in a sample from an individual using at least one anti-Aβ fibrils / oligomery antibody or antibody fragment as disclosed herein, wherein the sample may be a soluble fraction or a fixed tissue sample.

[0024] This disclosure provides a method for screening for at least one symptom associated with Aβ protein aggregation in an individual by using an anti-Aβ fibril / oligomer antibody or antibody fragment as disclosed herein to detect Aβ fibrils / oligomers in a sample from the individual.

[0025] This disclosure provides a method for monitoring at least one symptom associated with Aβ protein aggregation in an individual, the method comprising detecting Aβ fibrils / oligomers in a sample from the individual using at least one anti-Aβ fibrils / oligomer antibody or antibody fragment as disclosed herein.

[0026] This disclosure provides an in vivo, in vitro, or ex vivo method for detecting soluble Aβ fibrils / oligomers in a sample by contacting a sample with at least one anti-Aβ fibrils / oligomer antibody or antibody fragment as disclosed herein and detecting the binding of the anti-Aβ fibrils / oligomer antibody or antibody fragment to Aβ fibrils / oligomers to indicate the presence of soluble Aβ fibrils / oligomers in the sample. This disclosure also provides an in vivo, in vitro, or ex vivo method for detecting soluble Aβ fibrils / oligomers in a sample by contacting a sample with at least one anti-Aβ fibrils / oligomer antibody or antibody fragment as disclosed (wherein the Aβ peptide-binding polypeptide is part of a fusion protein or conjugate) and detecting the binding of the anti-Aβ fibrils / oligomer antibody or antibody fragment to Aβ fibrils / oligomers to indicate the presence of soluble Aβ fibrils / oligomers in the sample.

[0027] This disclosure provides an in vivo, in vitro, or ex vivo method for reducing the amount of soluble Aβ fibrils / oligomers in a sample suspected of containing Aβ fibrils / oligomers, the method comprising contacting the sample with an effective amount of at least one anti-Aβ fibrils / oligomer antibody or antibody fragment as disclosed herein to form a mixture, and then recovering the anti-Aβ fibrils / oligomer antibody or antibody fragment from the mixture, thereby removing Aβ fibrils / oligomers bound to the anti-Aβ fibrils / oligomer antibody or antibody fragment from the sample.

[0028] This disclosure provides a method for generating an antibody or antibody fragment against Aβ fibrils / oligomers, said antibody or antibody fragment preferentially binding to soluble Aβ fibrils / oligomers and having a low risk of immunogenicity, i.e., a low risk of adverse reactions or other unwanted side effects when administered to an individual, particularly when administered to humans. The currently disclosed method includes selecting a mammal capable of producing antibodies with a fully human variable region, immunizing the mammal by introducing a formulation of purified Aβ fibrils into the mammal, collecting enriched B cells from the spleen and lymph nodes of the mammal immunized with the formulation of purified Aβ fibrils, fusing the B cells with a myeloma fusion couple to generate a hybridoma, recovering antibodies from each of the hybridomas, performing an initial screening of the antibodies recovered from each of the hybridomas (including measuring binding to the Aβ peptide in an ELISA-based assay, identifying and recovering any antibody with a sufficient level of detectable binding to the Aβ peptide), performing a confirmatory screening of each recovered antibody after the initial screening (including measuring the competition between Aβ monomers and Aβ fibrils binding to the recovered antibody using a competitive ELISA), and measuring the direct binding of each recovered antibody to the Aβ monomer, Aβ fibrils, and Aβ filaments by an ELISA, and the method further includes selecting each recovered antibody that shows a higher affinity for fibrils than for monomers in a competitive ELISA.

[0029] This disclosure provides an isolated nucleic acid molecule that encodes at least a portion of an anti-Aβ fibril / oligomer antibody or antibody fragment as disclosed herein, wherein the nucleotide sequence of the isolated nucleic acid molecule comprises at least 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical nucleotide sequences to any one of the following: SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:40, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:60, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:75, SEQ ID NO:80, SEQ ID NO:85, SEQ ID NO:90, SEQ ID NO:95, SEQ ID NO:100, SEQ ID NO:105, SEQ ID NO:1 ... SEQ ID NO:115, SEQ ID NO:120, SEQ ID NO:125, SEQ ID NO:130, SEQ ID NO:135, SEQ ID NO:140, SEQ ID NO:145 and SEQ ID NO:150. This disclosure provides a vector comprising an isolated nucleic acid molecule encoding at least a portion of an anti-Aβ fibril / oligomer antibody or antibody fragment as disclosed herein. Attached Figure Description

[0030] Figure 1 This shows the reason for recycling Aβ 1-42 The purpose of the purified formulation of protofibrils was to detect Aβ by size exclusion chromatography (SEC). 1-42 The conformational analysis was performed. Aβ was... 1-42 The monomer solution was incubated at 37°C for 2 hours to form Aβ. 1-42 A mixture of fibrils and monomers. 200Increase 10 / 300GL column on-board analysis of Aβ via SEC 1-42 Control solutions of monomers (dashed lines) and Aβ1-42 fibrils and monomer mixtures (solid black lines). Figure 1 Aβ was shown 1-42 Monomer (dashed line) control sample and Aβ 1-42 Superposition of SEC results for fibrils and monomer mixtures (solid black line), where Aβ in each sample 1-42 fibrils and Aβ 1-42 Area % of single-unit peaks. Collected from Aβ1-42 The sample of the fibril peak was used as purified Aβ. 1-42 Fibrous fibers are used for immunity.

[0031] Figure 2A-2B The results of direct ELISA using reference antibodies rfmAb-1 and rfmAb-2 relative to different Aβ conformations are shown. The Aβ monomer (Aβ...) 1-40 monomers), Aβ fibrils (Aβ) 1-42 (fiber) or Aβ fiber (Aβ) 1-42 The filaments were coated onto an ELISA plate at 50 pmol / well. Diluted solutions of rfmAb-1 and rfmAb-2 were applied to the plate, and the binding of the antibody to Aβ in each well was detected by HRP-labeled secondary antibody and TMB substrate. Figure 2A As measured by direct ELISA, rfmAb-1 is effective against Aβ monomers (Aβ). 1-40 EC (monomer, hollow circle) 50 The value is 0.03 nM, and rfmAb-1 is effective against Aβ fibrils (Aβ). 1-42 EC (fiber, hollow square) 50 The value is 0.04 nM, and rfmAb-1 is effective against Aβ fibrils (Aβ... 1-42 EC (fiber, hollow triangle) 50 It is 1.1 nM. Figure 2B As measured by direct ELISA, rfmAb-2 is effective against Aβ monomers (Aβ). 1-40 EC (monomer, hollow circle) 50 The concentration of rfmAb-2 is 0.05 mM, which affects Aβ fibrils (Aβ). 1-42 EC (fiber, hollow square) 50 The value is 0.05 nM, and rfmAb-2 is effective against Aβ fibrils (Aβ... 1-42 EC (fiber, hollow triangle) 50 It is 0.08 nM.

[0032] Figures 3A-3B The results of a competitive ELISA of reference antibodies rfmAb-1 and rfmAb-2 relative to different Aβ conformations are shown. Each antibody (0.12 nM for rfmAb-1 and 0.21 nM for rfmAb-2) was mixed with the Aβ monomer (Aβ... 1-40 monomers), Aβ fibrils (Aβ) 1-42 (fiber) or Aβ fiber (Aβ) 1-42 The antibody-Aβ mixture was pre-incubated with the diluent (filaments) for 1 hour. Then, the antibody-Aβ mixture was applied to an ELISA plate coated with Aβ monomers for 10 minutes, and binding to the Aβ monomers was detected by HRP-labeled secondary antibody and TMB substrate. Figure 3A As calculated, Aβ monomer (Aβ 1-40 Monomer, hollow circle) IC for rmAb-1 50 The Aβ fibril has a molecular weight of 991.3 nM. 1-42 fibrils (hollow square) for rmAb-1 IC 50 It is 1.0 nM, and Aβ filaments (Aβ 1-42 Fibers (hollow triangles) for IC of rfmAb-1 50 It is 128.7 nM. Figure 3B As calculated, Aβ monomer (Aβ 1-40 Monomer, hollow circle) IC of rfmAb-2 50 It is 1.5 nM, Aβ fibrils (Aβ 1-42 fibrils (hollow square) for IC of rfmAb-2 50 It is 5.4 nM, and Aβ filaments (Aβ 1-42 Fibers (hollow triangles) for IC of rfmAb-2 50 It is 179.5 nM.

[0033] Figure 4 The results of capture ELISA using reference antibodies rfmAb-1 and rfmAb-2 are shown. Each antibody (100 ng / well) was coated onto a 96-well plate. Biotin-conjugated Aβ was then added. 40 The monomer diluent was applied to the plate and left for 1 hour, and binding was detected by HRP-conjugated streptavidin and TMB substrate. EC50 of rfmAb-1 (solid circle) 50 It cannot be reliably measured and is considered unstable. EC of rfmAb-2 (solid square) 50 It is 0.067 nM.

[0034] Figures 5A-5B Results for five leader antibodies (18P01A, 17P04A, 20O07A, 22D04A, and 06E17A) and the reference antibody rfmAb-1 in direct ELISA are shown. Aβ 1-42 ( Figure 5A ) or Aβ p3-42 ( Figure 5B The antibody was coated at 50 pmol / well onto the wells of an ELISA plate. Then, the antibody diluent was applied to the plate. Binding was detected using HRP-labeled secondary antibody and TMB substrate. Figure 5A The antibody and Aβ were shown. 1-42The direct bonding of fibrils, 18P01A (hollow circle), 17P04A (hollow square), 20O07A (upward-pointing triangle), 22D04A (downward-pointing triangle) and 06E17A (hollow rhombus) EC 50 The values ​​are 0.062 nM, 0.045 nM, 0.050 nM, 0.035 nM and 0.052 nM, respectively. Figure 5B The antibody and Aβ were shown. p3-42 The combination of these antibodies, in which EC50 of all lead antibodies cannot be measured, is problematic. 50 This indicates that it is related to Aβ p3-42 The binding is weak or nonexistent.

[0035] Figures 6A-6E The results for five leader antibodies in a competitive ELISA are shown. Each antibody was mixed with an Aβ monomer (Aβ... 1-40 monomers), Aβ fibrils (Aβ) 1-42 (fiber) or Aβ fiber (Aβ) 1-42 The antibody-Aβ mixture was pre-incubated with the diluent for 1 hour. Then, the antibody-Aβ mixture was plated onto an ELISA plate coated with Aβ monomers and incubated for 10 minutes. Binding was detected using HRP-labeled secondary antibody and TMB substrate. The Aβ monomer (Aβ...) is shown in the image. 1-40 Monomer, hollow circle), Aβ fibrils (Aβ) 1-42 Protofibrils, hollow squares), Aβ filaments (Aβ) 1-42 The results for each antibody (filaments, hollow triangles), of which Figure 6A The results for antibody 18P01A are shown. Figure 6B The results for antibody 17P04A are shown. Figure 6C The results for antibody 20O07A are shown. Figure 6D The results for antibody 22D04A were shown and Figure 6E The results for antibody 06E17A are shown.

[0036] Figure 7 The results show the capture of soluble biotin-Aβ monomers using immobilized antibody (0.5 μg / well) with each of the five leader antibodies: 18P01A (hollow circle), 17P04A (hollow square), 20O07A (upward-pointing triangle), 22D04A (downward-pointing triangle), and 06E17A (hollow rhombus), as well as two reference antibodies: rfmAb-1 (solid circle) with low affinity for the monomer and rfmAb-2 (solid square) with high affinity for the monomer.

[0037] Figure 8Results of ADCP assays using BV2 microglia are shown, with antibodies 18P01A (hollow circles), 17P04A (hollow squares), 22D04A (pointing triangles), rfmAb-1 (solid circles), rfmAb-2 (solid squares), and mouse IgG2b used as negative controls (hybridization). HiLyte488-labeled fibrils (2.25 μg / mL) were mixed with serial dilutions of the antibody (800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL) for 30 minutes. Each mixture was added to BV-2 cells and incubated at 37°C for 1.5 hours to induce ADCP. Positive fluorescence signals from the ingested HiLyte488-labeled fibrils were analyzed by flow cytometry, and relative fluorescence values ​​were calculated.

[0038] Figure 9 Serum pharmacokinetic (PK) data for antibodies 17P04A (hollow squares), 18P01A (hollow circles), and rfmAb-1 (solid circles) in B6SJLF1 mouse serum are shown. Values ​​at each time point are averages from samples of 2–3 animals. The calculated PK parameters for each antibody in serum are also shown.

[0039] Figure 10 Brain PK data for antibodies 17P04A (hollow squares), 18P01A (hollow circles), and rfmAb-1 (solid circles) in B6SJLF1 mice are shown. Values ​​at each time point are averages from samples of 2–3 animals. Calculated PK parameters for each antibody in the brain are also shown.

[0040] Figure 11A-11B The following figures illustrate the RIPA-soluble fractions of brain homogenates from naïve (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice treated with PBS mediator (Veh) via weekly intraperitoneal injection, 11-month-old 5xFAD mice treated with rfmAb-1 via weekly intraperitoneal injection of 1 mg / kg, 3 mg / kg, or 10 mg / kg for 16 weeks, and 11-month-old 5xFAD mice treated with 17P04A via weekly intraperitoneal injection of 1 mg / kg, 3 mg / kg, or 10 mg / kg for 16 weeks. Figure 11A ) and guanidine soluble fraction ( Figure 11B Aβ in ) 1-42 Level. N = 13, 14, 8, 8, 10, 8, 9, 9. In Figure 11BIn the results, the one-way ANOVA was followed by the Sidak test: ** indicates p < 0.01 for 3 mg / kg or 10 mg / kg rfmAb-1 against PBS mediator (Veh); **** indicates p < 0.0001 for 1 mg / kg, 3 mg / kg or 10 mg / kg 17P04A against PBS mediator (Veh); #p < 0.05 for 3 mg / kg rfmAb-1 against 3 mg / kg 17P04A; ##p < 0.01 indicates 10 mg / kg rfmAb-1 against 10 mg / kg 17P04A.

[0041] Figure 12A-12B The following figures illustrate the RIPA-soluble fractions of brain homogenates from naïve (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice treated with PBS mediator (Veh) via weekly intraperitoneal injection, 11-month-old 5xFAD mice treated with rfmAb-1 via weekly intraperitoneal injection of 1 mg / kg, 3 mg / kg, or 10 mg / kg for 16 weeks, and 11-month-old 5xFAD mice treated with 17P04A via weekly intraperitoneal injection of 1 mg / kg, 3 mg / kg, or 10 mg / kg for 16 weeks. Figure 12A ) and guanidine soluble fraction ( Figure 12B The Aβ1-40 levels in (N = 13, 14, 8, 8, 10, 8, 9, 9). Figure 12B In the results, the single-phase ANOVA was followed by the Sidak test: **for 1 mg / kg rfmAb-1 mediator (Veh), p < 0.01.

[0042] Figure 13 The levels of Aβ oligomers / fibrils in the TBS soluble fractions of brain homogenates from naïve (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice treated with PBS mordant (Veh) via weekly intraperitoneal injection, 11-month-old 5xFAD mice treated with rfmAb-1 via weekly intraperitoneal injection of 1 mg / kg, 3 mg / kg, or 10 mg / kg for 16 weeks via weekly intraperitoneal injection of 17PO4A via weekly intraperitoneal injection of 1 mg / kg, 3 mg / kg, or 10 mg / kg for 16 weeks via weekly intraperitoneal injection of 17PO4A are shown. Aβ oligomer levels were measured using a sandwich ELISA, where the capture Ab was rfmAb-1 and the detection Ab was biotin-18PO1A. N = 13, 14, 8, 8, 10, 8, 9, and 9. The results of the one-way ANOVA were then obtained from the Sidak test: ***for 10 mg / kg of 17P04 as a mediator (Veh), p < 0.001.

[0043] Figures 14A-14B The following figures show serum Aβ levels from naïve (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice treated with weekly intraperitoneal injections of PBS mediator (Veh), 11-month-old 5xFAD mice treated with rfmAb-1 for 16 weeks via weekly intraperitoneal injections of 1 mg / kg, 3 mg / kg, or 10 mg / kg, and 11-month-old 5xFAD mice treated with 17PO4A for 16 weeks via weekly intraperitoneal injections of 1 mg / kg, 3 mg / kg, or 10 mg / kg, 17PO4A. 1-42 level( Figure 14A ) and Aβ 1-40 level( Figure 14B ). Figure 14A N = 6 - 10; Figure 14B N = 5-13. In Figure 14A In the meantime, the results of the one-way ANOVA followed by the Sidak test: *for 10 mg / kg of 17P04 as a mediator (Veh), p < 0.05.

[0044] Figures 15A-15B Campbell-Switzer staining of brain sections from 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice treated with weekly intraperitoneal injections of PBS mediator (Veh), 11-month-old 5xFAD mice treated with rfmAb-1 for 4 months by weekly intraperitoneal injections of 1 mg / kg, 3 mg / kg, or 10 mg / kg, and 11-month-old 5xFAD mice treated with 17P04A for 4 months by weekly intraperitoneal injections of 1 mg / kg, 3 mg / kg, or 10 mg / kg, are shown. Figure 15A ) and the calculated amounts of diffuse and condensed Aβ plaques in the brain ( Figure 15B ). Figure 15A A representative image of Campbell-Switzer staining on a brain slice located approximately 1.30 mm from the anterior fontanelle. Figure 15B The calculated percentage of area of ​​condensed Aβ plaques (top) and diffuse Aβ plaques (bottom) in the brains of WT(B6SJLF1 / J) and 5xFAD mice; the percentage of area in each brain is the average of four brain slices at -0.25 mm, -1.30 mm, -2.35 mm, and -3.40 mm of the anterior fontanelle. n = 10, 13, 8, 8, 10, 8, 9, 9. Figure 15BIn the results, the one-way ANOVA was followed by the Sidak test: **p<0.01 against the mediator (Veh); ***p<0.001 against the mediator (Veh); at the same dose, #p<0.05rfmAb-1 against 17PO4; at the same dose, ##p<0.01rfmAb-1 against 17PO4. Detailed Implementation

[0045] This disclosure relates to and provides novel anti-Aβ fibrils / oligomery antibodies and antibody fragments that preferentially bind to soluble Aβ fibrils / oligomers and have a fully human variable region, wherein the anti-Aβ fibrils / oligomery antibodies and antibody fragments can trigger antibody-dependent cell-mediated phagocytosis (ADCP) in cells including microglia exposed to the anti-Aβ fibrils / oligomery antibodies and antibody fragments bound to Aβ fibrils / oligomers, and additionally, wherein the anti-Aβ fibrils / oligomery antibodies and antibody fragments can penetrate brain tissue after administration to a mammalian individual and reduce the levels of soluble Aβ oligomers / fibrils and insoluble Aβ fibrils / plaques, including concentrated plaques and diffuse plaques, in the brain tissue measured after administration to the individual. It should be noted that both Aβ oligomers and Aβ fibrils are soluble neurotoxic Aβ aggregates formed from monomers, presumably in different structures and molecular weights, but not necessarily bound to a specific model or mechanism of action. This disclosure relates to and provides novel anti-Aβ fibril / oligomer antibodies and antibody fragments that preferentially bind to soluble Aβ fibrils / oligomers and provide therapeutic effects associated with reducing the levels of soluble Aβ oligomers / fibrils in one or more tissues after administration to an individual.

[0046] Terms / Definitions

[0047] Unless otherwise defined, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Unless the context clearly requires otherwise, the use of singular terms (“a or an” or “the” or other uses of the singular form of the term) includes plural referents, and plural terms shall include the singular. Thus, for example, reference to “antibody” includes “one or more” antibodies or “multiple” such antibodies. All publications mentioned herein are hereby incorporated in their entirety by reference.

[0048] Generally, terminology and techniques of molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry, and recombinant DNA technology known to those skilled in the art can be used for the antibodies, antigen-binding fragments, compositions, and methods disclosed herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references, particularly Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Ausubel et al. (1994), *Current Protocols in Molecular Biology*, Volumes I-III (John Wiley & Sons, NY). Unless otherwise specified herein, enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. This article uses standard terminology to describe the techniques and methods used for drug preparation and formulation, as well as individualized treatment.

[0049] In the broadest sense, “antibody” and “antibody fragment” refer to a polypeptide or combination of polypeptides that recognizes and binds to an antigen via one or more immunoglobulin variable regions, which may be naturally occurring or non-natural, for example, as a result of engineering, chimerism, humanization, optimization, CDR transplantation, or affinity maturation. Antibodies or antibody fragments disclosed herein contain at least sufficient complementarity-determining regions (CDRs) interspersed with frame regions (FRs) for antibody recognition and binding to antigens. Depending on the desired function, antibodies or antibody fragments disclosed herein may include additional peptides or other portions, such as sufficient constant region (also called constant domain) structures / sequences to elicit a desired response, such as ADCPs in effector cells, or regions supporting labeling, tagging, linkers, conjugation, etc., to allow said antibody or antibody fragment to be detected, imaged, conjugated, labeled, crosslinked, immobilized, removed, etc.

[0050] Antibodies or antibody fragments can be, but are not limited to, at least one of the following: monoclonal antibodies, recombinant monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, single-chain variable fragments (scFv), aptamers, single-domain antibodies (VHH or nanobodies), recombinant antibodies, modified antibodies having a peptide / other portion linked to the antibody and / or additional amino acids added to an N-terminus or C-terminus, or other antigen-binding fragments or variants. The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain (HC) and / or light chain (LC) is derived from a specific source or species, while the remaining portions of the HC and / or LC are derived from different sources or species. For example, the target binding region (typically, the variable region) will be from one species (e.g., human, non-human primate, or mouse) and the constant region (especially the Fc) will be from a different species. Chimeric antibodies can be generated from sources engineered to produce antibodies having defined variable and defined constant regions, for example, from mice engineered to express whole-human variable immunoglobulin regions to produce antibodies having both whole-human variable and constant regions (which may include mouse constant region sequences, such as mouse Fc), and / or from cells engineered to produce antibodies having similar defined variable / constant region forms. It should be understood that this disclosure relates to providing antibodies having whole-human variable regions, wherein in some embodiments, the antibody may be a naturally occurring chimeric antibody generated from cells of mice (Musmusculus) engineered to produce antibodies having whole-human variable regions. It should be understood that in other embodiments, the antibody may be a recombinant antibody generated from cells expressing one or more constructs encoding antibodies having whole-human variable regions in a defined variable / constant region form, such as whole-human antibodies or chimeric antibodies, including a constant region component of the Fc derived from a class / subclass (isotype) selected for a particular use and the intended target of the antibody. As used herein, the phrase “humanized antibody” refers to an antibody or antibody variant derived from a non-human antibody having a non-human variable region, typically a mouse monoclonal antibody, in which a CDR derived from a parental non-human antibody is transplanted (fused) into a frame containing a variable region derived from a human immunoglobulin frame, particularly a recipient human frame or a human shared frame.The techniques and principles used for designing, preparing, and testing humanized antibodies are known (Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986 May 29–June 4; 321(6069):522-5; Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 2008 Jan 1; 13:1619-33). It should be understood that the receptor framework can be modified at multiple sites to develop humanized antibodies with improved characteristics (e.g., high affinity for the target, low clearance, low toxicity, etc.) for the desired use. The constant regions (constant domains) of antibodies or antibody fragments can be derived from one of the five major classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM), and several of these antibodies can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, each of which is well-characterized and known to confer functional specialization. Modified versions of each of these classes and isotypes are readily identifiable and within the scope of this disclosure. While all immunoglobulin classes are within the scope of this disclosure, this disclosure will primarily relate to the IgG class of immunoglobulin molecules.

[0051] The anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein may be complete (full-length) antibodies, single-chain antibodies, or antibody fragments having one or two chains, and may be naturally occurring or non-natural. The antibodies or antibody fragments disclosed herein contain at least sufficient complementarity-determining regions (CDRs) interspersed with frame regions (FRs) for antibody recognition and binding to antigens, preferably Aβ fibrils / oligomers. The anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein may have a partially or completely variable region portion comprising at least a sufficient amount of a fully human heavy chain variable region polypeptide (VH) and a sufficient amount of a fully human light chain variable region polypeptide (VL), which together form a structure having a binding domain that interacts with the antigen, preferably soluble Aβ fibrils / oligomers, and it should be understood that the variable region of the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein may include two VH-VL structures. As disclosed herein, the anti-Aβ fibril / oligomery antibody can be a full-length antibody, an intact antibody, a naturally occurring antibody, or equivalent terms. These terms should be understood to refer to a polypeptide comprising two full-length heavy chains (HC) and two light chains (LC) linked together by disulfide bonds to form a constant region and a variable region. It should be understood that the anti-Aβ fibril / oligomery antibody or antibody fragment disclosed herein, particularly the anti-Aβ fibril / oligomery antibody fragment disclosed herein, may contain HC polypeptides that are not necessarily considered full-length HC, especially in the constant region, but have sufficient structures / sequences for the desired function, such as constant region structures / sequences sufficient to elicit the desired response, particularly Fc structures / sequences sufficient to trigger ADCP in effector cells, or constant region structures / sequences sufficient to support labeling, tagging, linker, conjugation, etc., to allow the antibody or antibody fragment to be detected, imaged, labeled, conjugated, cross-linked, immobilized, removed, etc.

[0052] In the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein, each VH and VL region can be further subdivided into CDR regions characterized by hypervariable regions interspersed with typically more conserved FR regions. Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system and the classical complement system) and / or can trigger function in effector cells. Typically, the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein contain at least the heavy chain (HC)-CDR1, HC-CDR2, and HC-CDR3 and the light chain (LC)-CDR1, LC-CDR2, and LC-CDR3 sequences. Tables 2 (VH) and 3 (VL) disclose the VH and VL amino acid sequences of non-limiting embodiments of anti-Aβ fibril / oligomer antibody or antibody fragments, wherein the CDRs identified using IMGT numbers (imgt.org) are indicated by underlining in each VH and VL region and provided as separate sequences. The sequence listings incorporated herein by reference disclose the nucleotide sequences of VH and VL encoding non-limiting embodiments of anti-Aβ fibril / oligomer antibody or antibody fragments. Antibodies may contain fewer CDR sequences, as long as the antibody can recognize and bind to the antigen.

[0053] The anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein may be variants containing at least one altered CDR or framework sequence, wherein the CDR and / or framework sequence can be optimized by mutating nucleic acid molecules encoding such framework sequences. The anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein may have HC and LC moieties independently derived from different sources. Techniques for generating variants include, but are not limited to, conserved amino acid substitutions, computer modeling, screening candidate peptides alone or in combination, and codon optimization, and it should be understood that those skilled in the art can generate antibody variants as needed. The anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein may be fragments, provided that said fragment retains the ability to trigger ADCP when bound to Aβ fibrils / oligomers. Antigen-binding function of an antibody can be achieved through the following fragments: Fab fragments; monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments; bivalent fragments comprising two Fab fragments linked at the hinge region by disulfide bonds; Fd fragments consisting of VH and CH1 domains; single-chain variable fragments (scFv) consisting of VL and VH domains of a single arm of the antibody; single-domain antibody (dAb) fragments consisting of VH domains; and isolated CDRs (VHH, nanobodies) or aptamers. The antigen-binding portion can be incorporated into single-domain antibodies, large antibodies, micro antibodies, nanobodies, intracellular antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of an antibody can be grafted into a peptide-based scaffold to form a monoclonal antibody (see, for example, U.S. Patent No. 6,703,199, which describes a fibronectin peptide monoclonal antibody).

[0054] Terms including “symptoms associated with Aβ protein aggregation” or “symptoms associated with Aβ protein aggregation and deposition” or “Aβ peptide-related symptoms” should be understood to refer to a group of symptoms, diseases and conditions associated with Aβ plaque formation, particularly extracellular Aβ plaque formation. Neurological symptoms associated with Aβ protein aggregation and deposition include, but are not limited to, Alzheimer's disease (AD), mild cognitive impairment (MCI), Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis and other diseases based on or associated with amyloid-like protein (such as cerebral amyloid angiopathy, primary and secondary generalized amyloidosis, familial amyloid polyneuropathy), progressive supranuclear palsy, multiple sclerosis, Creutzfeld-Jacob disease, Parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), type II diabetes and age-related cardiac amyloidosis; as well as various eye diseases, including glaucoma, macular degeneration, drusen-related optic neuropathy and cataracts due to β-amyloid protein deposition.

[0055] In this disclosure, the use of terms including “Aβ fibrils / oligomers” and “Aβ fibrils and / or oligomers Aβ” and similar language should be understood as meaning that Aβ oligomers and Aβ fibrils are considered to be soluble neurotoxic Aβ formed from monomers and are expected to be present in the sample, presumably in a variety of different structures, conformations, associations, and molecular weights (e.g., fibrils > 75 kDa and oligomers < 75 kDa), such that “Aβ fibrils / oligomers” can encompass Aβ fibrils, or Aβ oligomers, or mixtures of Aβ fibrils and Aβ oligomers, or complexes (associations) of Aβ fibrils and Aβ oligomers. Therefore, terms such as “detection of Aβ fibrils / oligomers” should be understood to encompass the detection of any or all of Aβ fibrils, or Aβ oligomers, or both Aβ fibrils and Aβ oligomers, or complexes (associations) of Aβ fibrils and Aβ oligomers.

[0056] In this disclosure, the phrase "preferentially binds to Aβ fibrils / oligomers" or "preferentially binds to soluble Aβ fibrils / oligomers" or similar language should be understood to indicate selectivity or affinity for Aβ fibrils / oligomers, which can be measured by commonly used methods known in the art, including those described herein. Approximately 50% maximum binding (calculated EC50) 50The calculated concentration at the time of occurrence can often be used as an estimate of affinity. Because Aβ protein in a sample can exist in various conformations of Aβ secondary structures (monomers, oligomers / fibrils, fibrils, etc.), it should be understood that "preferential binding" or "selectivity," or similar language, means that the binding of an antibody or antibody fragment to Aβ oligomers / fibrils is measurably higher than the binding of the same antibody or antibody fragment to Aβ monomers in the sample. A non-limiting method for determining the selectivity of Aβ oligomers / fibrils to Aβ monomers can be calculated as EC measured in a capture ELISA assay. 50 IC50 measured in monomeric and competitive ELISA assays 50 The ratio of oligomers. This ratio is used to identify and classify antibodies and antibody fragments against Aβ fibrils / oligomers based on the level of preferential binding to soluble Aβ oligomers / fibrils.

[0057] "Individual" is a mammal, including but not limited to primates (e.g., humans and non-human primates such as monkeys), mammals commonly used for research such as rabbits and rodents (e.g., mice and rats), and domesticated animals (e.g., cattle, sheep, cats, dogs, pigs, llamas, and horses). In some embodiments, the individual is a person. The phrases "to individuals in need," "to patients in need," "to patients in need of treatment," or "individuals in need of treatment" can include individuals who will benefit from administration of the anti-Aβ fibrils / oligomery antibodies and antibody fragments disclosed herein for the treatment, prevention, diagnosis, screening, or monitoring of symptoms associated with Aβ protein aggregation, particularly AD. It should be understood that administration of anti-Aβ fibrils / oligomery antibodies or antibody fragments covering administration to "individuals in need" can be interpreted as referring to individuals known or suspected of having symptoms associated with Aβ protein aggregation, particularly AD. The anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein can be administered to individuals with known or suspected conditions associated with Aβ protein aggregation, particularly Alzheimer's disease (AD), for therapeutic, preventative, or control purposes, including but not limited to treatment, screening, diagnosis, surveillance, research, or achieving outcomes different from those for treating the condition. It should be further understood that anti-Aβ fibrils / oligomery antibodies or antibody fragments can be administered to individuals with unknown or suspected conditions associated with Aβ protein aggregation for purposes including but not limited to prevention or control, screening, diagnosis, surveillance, research, or achieving outcomes different from those for treating the condition.

[0058] Composition

[0059] Anti-Aβ fibrils / oligomery antibodies and antibody fragments that exhibit selectivity for and preferential binding to soluble Aβ fibrils / oligomery compounds, and trigger ACDP in effector cells, penetrate brain tissue, reduce the level of soluble Aβ fibrils / oligomery compounds in brain tissue, reduce insoluble Aβ plaques in brain tissue, including condensed and diffuse plaques, and reduce plaque burden.

[0060] Antibody-antigen binding fragments exhibiting selectivity for and preferential binding to soluble Aβ fibrils / oligomers are provided. Specifically, anti-Aβ antibody-antigen binding fragments with sufficient constant region sequences including sufficient Fc structures / sequences are provided such that a complex of anti-Aβ fibrils / oligomer antibody or antibody fragments bound to Aβ fibrils / oligomers can trigger ADCP in effector cells with phagocytic potential, thereby generating phagocytosis and thus removing the complex of anti-Aβ fibrils / oligomer antibody or antibody fragments bound to Aβ fibrils / oligomers from the solution surrounding the effector cells. Without being bound by a specific mechanism of action or assumption, ADCP-mediated removal of the complex of anti-Aβ fibrils / oligomer antibody or antibody fragments bound to Aβ fibrils / oligomers from the solution surrounding the effector cells can achieve the removal of soluble Aβ fibrils / oligomers from said solution. It should be understood that ADCP-mediated removal of anti-Aβ fibrils / oligomery antibody or antibody fragment complexes bound to Aβ fibrils / oligomers from the solution surrounding effector cells can occur in vivo, in vitro, or ex vivo. In vivo ADCP-mediated removal of these complexes can occur particularly in blood, lymph, cerebrospinal fluid (CSF), neural tissue, or the brain. Therefore, in vivo ADCP-mediated removal of these complexes can reduce soluble Aβ fibrils / oligomery antibody or antibody fragment levels, particularly in blood, lymph, CSF, neural tissue, or the brain. The removal of complexes of anti-Aβ fibrils / oligomers or antibody fragments bound to Aβ fibrils / oligomers, either in vitro or in vitro, mediated by ADCP, can occur in samples collected from individuals (such as blood, lymph, cerebrospinal fluid (CSF)) or other samples, and can reduce the level of soluble Aβ fibrils / oligomers in such samples.

[0061] As presented in this article, anti-Aβ antibodies with fully human variable regions and antigen-binding fragments carry a lower risk of inducing an immune response, and when administered to human individuals, a reduced risk of adverse reactions or unwanted side effects is expected. In contrast, humanized antibodies derived from non-human antibodies, such as lencanezumab, a humanized IgG1 monoclonal antibody that preferentially binds to Aβ fibrils / oligomers, can be expected to carry a relatively higher risk of adverse reactions or unwanted side effects after administration.

[0062] Exemplary embodiments of antibodies and antigen-binding fragments that preferentially bind to soluble Aβ fibrils / oligomers and trigger ADCP include, but are not limited to, the antibodies provided in Examples and Tables 1, 2, 3, 4, and 5. In some embodiments, the antibodies obtained by using Aβ fibrils / oligomers are recovered from the hybridoma supernatant. 1-42 Transgenic mice immunized with fibrils produced anti-Aβ fibrils / oligomery antibodies with fully human variable regions and were screened as described in Example 1 to produce chimeric monoclonal antibodies with fully human F(ab')2 and mouse CH2 and CH3 domains. In some embodiments, the ability of thirty-three (33) strains of chimeric monoclonal antibodies with fully human F(ab')2 and mouse IgG1 CH2 and CH3 domains as listed in Table 1 to preferentially bind to soluble Aβ fibrils / oligomers and trigger ADCP was evaluated, and the VH and VL sequences of fifteen (15) antibodies of interest are provided in Tables 2 and 3. In some embodiments, anti-Aβ fibrils / oligomer antibodies are prepared in different forms for different practical and potential uses and their ability to preferentially bind to soluble Aβ fibrils / oligomers and trigger ADCP is evaluated, including: chimeric monoclonal antibodies having fully human F(ab')2 and mouse IgG1 CH2 and CH3 domains as shown in Table 1 (i.e., chimeric human F(ab')2 / mouse IgG1 antibodies), wherein the variable region sequences are presented in Tables 2 and 3; antibodies reformatted as fully human IgG1λ antibodies and tested as described in Example 5 and as shown in Table 4; and antibodies reformatted as mouse IgG2a CH2-CH3 / (fully human F(ab')2 and mouse IgG2a CH2 and CH3 domains) and tested as described in Example 5 and as shown in Table 5 and used as in Examples 6 and 7.

[0063] The sequences and forms of anti-Aβ fibrils / oligomer antibodies and antibody fragments can be modified to develop antibodies or fragments with desired properties. In one non-limiting embodiment, anti-Aβ fibrils / oligomer antibodies or antibody fragments with desired properties for a specific purpose can be developed using a suitable variable region framework in combination with a constant region sequence, the variable region framework comprising a VH containing HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO:42), HC CDR2 having the amino acid sequence ISSRTYI (SEQ ID NO:43), and HC CDR3 having the amino acid sequence ARGGYIGSPNAYDI (SEQ ID NO:44), and a light chain variable region (VL) containing LC CDR1 having the amino acid sequence TGAVTSDYY (SEQ ID NO:47), LC CDR2 having the amino acid sequence SAS, and LC CDR3 having the amino acid sequence LLYYGGAWV (SEQ ID NO:49), the constant region sequence including an Fc sequence selected to be suitable for the specific purpose. In another non-limiting embodiment, an anti-Aβ fibril / oligomer antibody or antibody fragment having desired properties for a particular purpose can be developed using a suitable variable region framework combined with a constant region sequence, the variable region framework comprising a VH containing HC CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO: 62), HC CDR2 having the amino acid sequence IRSKANSYAT (SEQ ID NO: 63), and HC CDR3 having the amino acid sequence TSHAPNFDAFDI (SEQ ID NO: 64), and a VL containing LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 67), LCCDR2 having the amino acid sequence DNS, and LC CDR3 having the amino acid sequence GTWDSSLSTYV (SEQ ID NO: 69), the constant region sequence including an Fc sequence selected to be suitable for the particular purpose.In another non-limiting embodiment, an anti-Aβ fibril / oligomer antibody or antibody fragment having desired properties for a particular purpose can be developed using a suitable variable region framework combined with a constant region sequence, the variable region framework comprising a VH containing HC CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO:72), HC CDR2 having the amino acid sequence IRSKVNSYAT (SEQ ID NO:73), and HC CDR3 having the amino acid sequence TSHAPIFDAFDI (SEQ ID NO:74), and a VL containing LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO:77), LC CDR2 having the amino acid sequence DNS, and LC CDR3 having the amino acid sequence GTWDSSLSTYF (SEQ ID NO:79), the constant region sequence including an Fc sequence selected to be suitable for the particular purpose. In another non-limiting embodiment, anti-Aβ fibrils / oligomery antibodies or antibody fragments having desired properties for a particular purpose can be developed using a suitable variable region framework combined with a constant region sequence, the variable region framework comprising a VH containing HC CDR1 having the amino acid sequence GFTFSNAW (SEQ ID NO: 132), HC CDR2 having the amino acid sequence IKSKTDGGTR (SEQ ID NO: 133), and HCCDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 134), and a VL containing LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO: 137), LC CDR2 having the amino acid sequence RNN, and LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 139), the constant region sequence including an Fc sequence selected to be suitable for the particular purpose.In another non-limiting embodiment, anti-Aβ fibrils / oligomery antibodies or antibody fragments having desired properties for a specific purpose can be developed using a suitable variable region framework combined with a constant region sequence, the variable region framework comprising a VH containing HC CDR1 having the amino acid sequence GFSFSNAW (SEQ ID NO:2), HC CDR2 having the amino acid sequence IKSKTDGGTI (SEQ ID NO:3), and HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO:4), and a VL containing LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO:7), LC CDR2 having the amino acid sequence RNN, and LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO:9), the constant region sequence including an Fc sequence selected to be suitable for the specific purpose. It should be understood that a “suitable variable region framework” is a suitable human variable region framework that can be selected or engineered for a specific property or purpose.

[0064] Anti-Aβ fibrils / oligomery antibodies and antibody fragments capable of penetrating brain tissue are provided. Specifically, anti-Aβ fibrils / oligomery antibodies and antibody fragments with fully human variable regions capable of penetrating brain tissue are provided. It should be understood that brain penetration, i.e., the ability to cross the blood-brain barrier, allows the antibody or antibody fragment to act on a target within brain tissue. Without being bound by a specific mechanism of action or assumption, ADCP-mediated removal of complexes of anti-Aβ fibrils / oligomery antibodies or antibody fragments bound to Aβ fibrils / oligomers in brain tissue may have effects that may include reducing the level of soluble Aβ fibrils / oligomers in brain tissue. Without being bound by a specific mechanism of action or assumption, ADCP triggered by target-bound anti-Aβ fibrils / oligomery antibodies or antibody fragments in brain tissue may have effects that may include reducing the amount of Aβ plaques in brain tissue. It should be understood that reducing the amount of Aβ plaques can encompass the removal of Aβ plaques from tissues and fluids.

[0065] Exemplary embodiments of the anti-Aβ fibrils / oligomery antibodies and antibody fragments tested as described in Example 6 can penetrate brain tissue, including but not limited to: 18P01A (mIgG2a) (HC amino acid sequence SEQ ID NO:155; LC amino acid sequence SEQ ID NO:156), including 18P01A full-human VH having the amino acid sequence SEQ ID NO:61 and 18P01A full-human VL having the amino acid sequence SEQ ID NO:66; and 17P04A (mIgG2a) (HC amino acid sequence SEQ ID NO:151; LC amino acid sequence SEQ ID NO:152, including 17P04A full-human VH having the amino acid sequence SEQ ID NO:41 and 17P04A full-human VL having the amino acid sequence SEQ ID NO:46). Both examples illustrate acceptable half-lives in serum after administration. Figure 9 And it is detectable in brain tissue after administration. Figure 10 Of these, 18P01A (mIgG2a) has a brain penetration of 0.3% and a half-life of 77.2 hours in brain tissue, and 17P04A (mIgG2a) has a brain penetration of 5.5% and a half-life of 92.4 hours in brain tissue. The serum half-life, brain penetration, and brain half-life of the additional anti-Aβ fibrils / oligomer antibodies and antibody fragments provided herein can be evaluated.

[0066] Anti-Aβ fibrils / oligomery antibodies and antibody fragments are provided that, upon administration to a mammalian individual, can reduce the levels of soluble Aβ oligomers / fibrils in the brain of that individual and reduce the levels of insoluble Aβ plaques (including condensed and diffuse plaques). Specifically, anti-Aβ fibrils / oligomery antibodies and antibody fragments having a fully human variable region are provided that, upon administration to a mammalian individual, can reduce the levels of soluble Aβ oligomers / fibrils in the brain of that individual and reduce the levels of insoluble Aβ plaques (including condensed and diffuse plaques). Without being bound by a specific mechanism of action or hypothesis, reducing the level of insoluble Aβ plaques in brain tissue can encompass reducing, preventing, or slowing the aggregation and deposition of Aβ proteins into insoluble Aβ plaques. Animal models of symptoms associated with Aβ protein aggregation were used to measure the effects of anti-Aβ fibrils / oligomery antibodies and antibody fragments on soluble Aβ oligomers / oligomers and to reduce the levels of insoluble Aβ plaques in brain tissue after administration. In a non-limiting exemplary embodiment using 5xFAD mice as an AD mouse model to evaluate the long-term efficacy of anti-Aβ fibrils / oligomery antibodies (Example 7), results showed that administration of 17PO4A (mIgG2a) reduced the levels of soluble Aβ oligomers / oligomers and insoluble Aβ plaques in the brains of 5xFAD mice compared to the levels of soluble Aβ oligomers / oligomers and insoluble Aβ plaques in control 5xFAD mice injected with the same amount of medulant (PBS) for the same duration. Figure 13 And reduce the level of insoluble Aβ plaques (including concentrated and diffuse plaques). Figures 15A-15B ). 17P04A (mIgG2a) (HC SEQ ID NO:151, LC SEQ ID NO:152) in a dose-dependent manner on soluble Aβ oligomers / fibrils ( Figure 13 ) and insoluble Aβ plaques ( Figure 15B This provides examples of how to assess parameters (such as antibody concentration, administration frequency, and duration of antibody treatment) to find optimal conditions that maximize therapeutic efficacy and avoid unwanted side effects. The effects of additional anti-Aβ fibrils / oligomery antibodies and antibody fragments provided here on soluble Aβ oligomers / fibrils and insoluble Aβ plaques in brain tissue can be evaluated after administration to an individual.

[0067] Compositions comprising the anti-Aβ fibrils / oligomery antibody or antibody fragment of the present invention and a pharmaceutically acceptable carrier or excipient suitable for the intended use of each composition are provided. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, excipients, stabilizers, preservatives, or combinations thereof. It should be understood that the pharmaceutical formulation should be matched to the mode of administration.

[0068] The anti-Aβ fibrils / oligomery antibodies and antibody fragments disclosed herein can be administered to an individual in any suitable manner, including but not limited to injection or parenteral administration. Parenteral administration may include intramuscular, intravenous, intra-arterial, intraperitoneal (“ip”) administration, subcutaneous administration, intraspinal (including epidural or intrathecal) administration, intraocular administration, intracerebral administration, intraventricular administration, intracardiac administration, intradermal / intradermal administration, intra-articular administration, intralymphatic administration, or intraosseous administration. This document discloses anti-Aβ oligomery antibodies and antibody fragments for introduction into the circulatory system via parenteral administration, particularly intraperitoneal administration. The anti-Aβ fibrils / oligomery antibodies and antibody fragments disclosed herein can be administered using a device, as a reservoir, or in the form of a sustained-release formulation (e.g., a semi-permeable matrix containing a solid hydrophobic polymer of the antibody, or microcapsules) to allow for slow and / or measurable and / or local delivery. The anti-Aβ fibrils / oligomery antibodies and antibody fragments disclosed herein can be formulated and administered using colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions.

[0069] method

[0070] Methods are provided for treating, preventing, diagnosing, screening, or monitoring conditions associated with Aβ protein aggregation, particularly Alzheimer's disease (AD), using effective amounts of the anti-Aβ fibrils / oligomery antibodies or antibody fragments disclosed herein. Without being bound by specific mechanisms of action, methods are provided for precisely targeting and inhibiting Aβ fibrils / oligomers, particularly soluble Aβ fibrils / oligomers, using the amounts and effects of the anti-Aβ fibrils / oligomery antibodies and antibody fragments disclosed herein. Methods for precisely targeting and inhibiting soluble Aβ fibrils / oligomers may include methods targeting the location or system involved in the disease using a route of administration, methods targeting key time periods using a timing of administration, and combinations of these methods.

[0071] Methods are provided for treating conditions associated with Aβ protein aggregation, particularly Alzheimer's disease (AD), by removing soluble Aβ fibrils / oligomers from tissues, fluids, or samples. Without being bound by a specific mechanism of action, methods are provided for treating conditions associated with Aβ protein aggregation by removing or reducing the level of Aβ protein aggregation precursors, particularly by ADCP-mediated removal of complexes of anti-Aβ fibrils / oligomer antibodies or antibody fragments bound to Aβ fibrils / oligomers. Methods and compositions are provided for treating conditions associated with Aβ protein aggregation, particularly AD, which can be used to remove soluble Aβ fibrils / oligomers from blood, lymph, cerebrospinal fluid (CSF), or from samples of interest collected from an individual. It should be understood that in vivo methods for treating conditions associated with Aβ protein aggregation involve administering a therapeutic amount of anti-Aβ fibrils / oligomer antibodies or antibody fragments to an individual.

[0072] The methods and compositions provided herein allow for strategic timing of treatment. Without being bound by a specific mechanism of action, the anti-Aβ fibrils / oligomer antibodies and antibody fragments disclosed herein can be administered after screening or diagnosis indicates that an individual has or is suspected of having symptoms associated with Aβ protein aggregation, particularly Alzheimer's disease (AD). Therefore, the anti-Aβ fibrils / oligomer antibodies and antibody fragments disclosed herein can be administered when the prevention or further development of symptoms associated with Aβ protein aggregation, particularly AD, can be prevented or potentially reversed. Without being bound by a specific mechanism of action, the anti-Aβ fibrils / oligomer antibodies and antibody fragments disclosed herein can be administered to individuals identified as being at risk of having or developing symptoms associated with Aβ protein aggregation, particularly AD. Therefore, the anti-Aβ fibrils / oligomer antibodies and antibody fragments disclosed herein can be administered when the onset or further development of symptoms associated with Aβ protein aggregation, particularly AD, can be prevented or prevented. Furthermore, the anti-Aβ fibrils / oligomer antibodies and antibody fragments disclosed herein can be administered preventively.

[0073] The methods and compositions provided herein allow for control of the amount of therapeutic or preventative agent present at one or more time points during treatment. For those who do not wish to be bound by a specific mechanism of action, the anti-Aβ fibrils / oligomer antibodies and antibody fragments disclosed herein can be administered, and effects, such as responses like edema or inflammation, indicators like soluble Aβ fibrils / oligomer levels or other Aβ conformations (monomers, filaments, plaques) levels, or clinical measurements of cognitive function can be monitored. For those who do not wish to be bound by a specific mechanism of action, the dose or “effective amount” sufficient to produce at least one desired effect (e.g., reduced soluble Aβ fibrils / oligomer levels, reduced Aβ plaque levels, or improved cognitive function) and few or tolerable undesirable effects (e.g., edema or inflammation) can depend on factors specific to the individual in need, such that treatment can comprise determining the dose of the anti-Aβ fibrils / oligomer antibody or antibody fragment required to constitute the effective amount for the individual and administering the effective amount of the anti-Aβ fibrils / oligomer antibody or antibody fragment to the individual.

[0074] A method is provided for diagnosing or screening an individual for at least one symptom associated with Aβ protein aggregation by using at least one anti-Aβ fibrils / oligomery antibody or antibody fragment as disclosed herein, to detect the presence and, optionally, the amount of soluble Aβ fibrils / oligomery in a sample from the individual. The sample may be a soluble fraction or a fixed tissue sample. It should be understood that the diagnostic or screening method involves comparing the result (such as the amount of soluble Aβ fibrils / oligomery in the sample) with at least one reference value associated with the presence and / or amount of soluble Aβ fibrils / oligomery in other samples. The reference value may be based on samples from other individuals (who may or may not be considered to have a symptom associated with Aβ protein aggregation, particularly AD) or values ​​from samples collected from the same individual at different time points.

[0075] A method is provided for generating anti-Aβ fibrils / oligomer antibodies or antibody fragments using a functional screening method, wherein the anti-Aβ fibrils / oligomer antibodies or antibody fragments preferentially bind to soluble Aβ fibrils / oligomers and have a fully human variable region. The method comprises immunizing a mammalian producing antibodies with a human variable region by introducing a formulation of purified Aβ fibrils (which may include oligomers) into the mammalian body; collecting enriched B cells and fusing the B cells with myeloma fusion couples to generate hybridomas; recovering antibodies from each of the hybridomas; performing an initial screening of the antibodies recovered from each of the hybridomas (including measuring binding to the Aβ peptide in an ELISA-based assay, identifying and recovering any antibody with a sufficient level of detectable binding to the Aβ peptide); performing a confirmatory screening of each recovered antibody after the initial screening (including measuring the competition between Aβ monomers and Aβ fibrils binding to the recovered antibody using a competitive ELISA); and measuring the direct binding of each recovered antibody to the Aβ monomer, Aβ fibrils, and Aβ filaments by an ELISA; and the method further comprises selecting each recovered antibody that shows a higher affinity for fibrils than for monomers in a competitive ELISA. It should be understood that mammals are engineered to produce antibodies with human variable regions, with mice typically engineered to achieve such functions. Each anti-Aβ fibril / oligomer antibody or antibody fragment can be formatted for a specific test or function, for example, by combining a constant region with a variable region (VH / VL), the constant region including an Fc region with desired function and / or species compatibility, and the variable region characterized as having a desired level of selectivity for and preferentially binding to soluble Aβ fibrils / oligomers. Additional screening and characterization steps are provided, including: measuring the ability of the anti-Aβ fibril / oligomer antibody or antibody fragment to trigger ADCP-mediated removal of complexes of the anti-Aβ fibril / oligomer antibody or antibody fragment bound to Aβ fibrils / oligomers; and assessing the brain penetration of the anti-Aβ fibril / oligomer antibody or antibody fragment and the subsequent reduction in plaque levels in brain tissue, thereby indicating the ability to clear plaques. The Fc region suitable for use with the anti-Aβ fibrils / oligomer antibodies or antibody fragments provided herein may be, but is not limited to, complete or partial Fc regions of mouse IgG1, human IgG1, mouse IgG2a, human IgG3 and mouse IgG2b.

[0076] In a non-limiting exemplary embodiment, results from long-term efficacy studies showed that treatment with the antibody efficiently and effectively removed soluble Aβ oligomers / fibrils and insoluble Aβ fibrils / plaques (including both condensed and diffuse plaques) from the brain, thereby reducing plaque burden in an AD disease model in vivo. The antibody has a fully human VH comprising 17P04A HC CDR1 having the sequence GFTLSSFS (SEQ ID NO:42), 17P04A HC CDR2 having the sequence ISSRRTYI (SEQ ID NO:43), and 17P04A HC CDR3 having the sequence ARGGYIGSPNAYDI (SEQ ID NO:44), and a fully human VL comprising 17P04A LCCDR1 having the sequence TGAVTSDYY (SEQ ID NO:47), 17P04A LCCDR2 having the sequence SAS, and LLYYGGAWV (SEQ ID NO:47). NO:49) 17P04A LCCDR3.

[0077] The following examples are provided to illustrate, rather than limit, the claimed invention.

[0078] Example

[0079] Example 1: Generation and identification of antibodies binding to amyloid-β (Aβ) oligomers / fibrils

[0080] The development of a novel monoclonal antibody against amyloid-beta (Aβ) oligomers was contracted to AlivaMab Discovery Services, LLC (San Diego, CA). AMX-KL mice (a transgenic mouse strain producing antibodies containing fully human F(ab')2 as well as mouse CH2 and CH3 domains, wherein fully human F(ab')2 comprises human VH and VL and human CL and CH1) were licensed from Ablexis, LLC (ablexis.com; San Diego, CA) and used for immunization to discover therapeutic antibodies. AMX-KL mice were immunized with freshly prepared Aβ fibrils. Aβ was prepared according to the protocol detailed below. 1-42 fibrils, and Figure 1Chromatographic curves are shown in Table 1. Enriched B cells with sufficient plasma titers from mouse spleen and lymph nodes were fused with myeloma fusion couples to trigger downstream antibody recovery and screening activities. Enzyme-linked immunosorbent assay (ELISA) was used for a series of screening and characterization steps. Initially, 15,662 hybridoma supernatants were screened for binding to the Aβ peptide in an ELISA-based assay. The top 186 hits with RLU > 6.9 million binding to the Aβ peptide were selected to advance to a confirmatory screening, which included measuring competition between Aβ monomers and fibrils as well as direct binding to monomers, fibrils, and filaments using ELISA. Thirty-three (33) hits from the confirmatory screening that showed a higher affinity for fibrils than monomers in a competitive ELISA were selected and purified. Following purification, each of the 33 monoclonal antibodies (mAbs) was characterized and ranked in a set of in vitro assays (results shown in Table 1), and the five (5) best-performing mAbs were identified as leader candidates. Fifteen (15) of the antibodies were subcloned to obtain variable region sequences (see Tables 2 and 3). Five (5) leader candidates were purified and characterized. The five leader candidates were then reformatted for further characterization and development into (a) fully human antibodies and (b) chimeric antibodies with different mouse isotypes.

[0081] Preparation of different Aβ conformations

[0082] 1mg Aβ 1-42 The monomer (rPeptide, A-1167-2; rpeptide.com) was dissolved in 200 μL of 12 mM NaOH. The solution was neutralized with 1200 μL of 10x PBS (final pH approximately 8.8) and incubated at 37 °C for 2 hours to allow fibrillation. Aβ was monitored by thiamine T assay (Sigma, T3516; sigmaaldrich.com; as a sample solution containing 10 μM thiamine T). 1-42 The aggregation state. This method produces a fibril / monomer mixture containing approximately 70% fibrils and approximately 30% monomers, such as... Figure 1 The results of size exclusion chromatography (Superdex 200Increase 10 / 300GL, Cytiva 28-9909-44) of the mixture are shown in the image. This fibril / monomer mixture was freshly prepared on each immunization day. Experiments were conducted based on the assumption that any Aβ in the mixture... 1-42 The monomer is unlikely to trigger an immune response due to its small size (MW 4514Da).

[0083] To prepare purified Aβ for antibody screening 1-42Fibrous fibers were collected, and fractions of the fibrillous fiber peaks were collected by size exclusion chromatography and stored at -80°C. This purified Aβ... 1-42 The fibrillated solution is stable at -80°C for at least 2 months and can withstand several freeze-thaw cycles. Aβ 1-42 Monomers are unstable and unsuitable for antibody screening. Therefore, Aβ, lacking two amino acid residues at the C-terminus, will be used. 1-40 The monomer (rPeptide, A-1157-2) is used as the monomer conformation for antibody screening. Aβ 1-40 The monomer is stable at 4°C for at least 24 hours and can be stored at -80°C for at least 2 months. Aβ 1-42 The filaments were purchased as preformed filaments from rPeptide (rPeptide Company, catalog number AF-1002).

[0084] Example 2: Characterization and sequencing of monoclonal antibodies against Aβ fibrils / oligomers

[0085] A series of ELISA-based binding assays, including direct ELISA, competitive ELISA, and capture ELISA, were developed for screening hybridomas and characterizing mAbs. Additionally, an antibody-dependent cell-mediated phagocytosis (ADCP) assay was developed to assess the potential of anti-Aβ antibodies to mediate the uptake of Aβ fibrils by microglia. Two control antibodies, anti-Aβ fibril / oligomery antibody reference antibody-1 (rfmAb-1) and non-conformation-selective anti-Aβ antibody 6E10 (BioLegend, 803001) (referred to as rfmAb-2), were used as references in those assays.

[0086] Direct ELISA

[0087] High-binding 96-well plates (Corning, 9018) were coated with 0.5 μM Aβ monomer or Aβ fibrils or Aβ fibers at 4°C for 2 hours. After washing three times with 1X phosphate-buffered saline and 0.1% Tween 20 (PBST), each plate was blocked at room temperature (RT) with PBS containing 1% BSA for 1 hour. Aβ antibody diluent (3-fold serial dilution in the range of 0-100 nM) was added to the plates. The plates were incubated at room temperature for 1 hour and washed three times. Then, the plates were incubated at room temperature with HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, G-21040; thermofisher.com) diluted 1:5000 in 1% BSA for 1 hour and washed three times. A TMB (3,3',5,5'-tetramethylbenzidine) stabilized chromogen substrate solution (Life Technologies (Thermo Fisher Scientific) SB02) was added to the plate, and the reaction was terminated by adding 1M sulfuric acid. The absorbance was measured at 450 nm using a microplate reader. In direct ELISA, the results for rfmAb-1 (oligomer / fibril-selective anti-Aβ antibody) and rfmAb-2 (non-conformation-selective anti-Aβ antibody) showed similar affinities for Aβ monomers and fibrils (Figure 2). This indicates that direct ELISA can measure the general affinity of antibodies for Aβ peptides but cannot determine the selectivity of the antibodies for different Aβ conformations.

[0088] Competitive ELISA

[0089] At 4°C, pre-incubate the Aβ antibody with Aβ monomers or Aβ fibrils or a diluent for Aβ fibrils (3-fold serial dilutions in the concentration range of 0-3000 nM) for 1 hour. The concentration of Aβ antibody used in the antibody-Aβ mixture is the EC90 of the antibody with Aβ monomers as measured in a direct ELISA assay. The mixture is then plated onto an ELISA plate pre-coated with 0.5 μM Aβ monomers. The plate is incubated at room temperature for 10 minutes and washed three times with TBST. The plate is then incubated at room temperature for 1 hour with HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, G-21040) diluted 1:5000 in 1% BSA and washed three times. TMB substrate solution (Lifetech, SB02) is added to the plate, and the reaction is terminated by adding 1M sulfuric acid. The absorbance is measured at 450 nm using a microplate reader. As shown in Figure 3, rfmAb-1 mixed with Aβ fibrils exhibits low binding with Aβ monomers, while rfmAb-1 mixed with Aβ monomers or fibrils exhibits high binding with Aβ monomers. Figure 3AThis indicates that rfmAb-1 has a high affinity for Aβ oligomers compared to Aβ monomers or fibrils, consistent with its known binding spectrum. As shown in Figure 3, in competitive ELISA, rfmAb-2 showed similar affinity for both Aβ monomers and Aβ fibrils. Figure 3B This is consistent with its known spectrum. These results indicate that competitive ELISA can distinguish the selectivity of anti-Aβ antibodies for different Aβ conformations.

[0090] Capture ELISA

[0091] A high-binding 96-well plate (Corning, 9018) was coated with 5 μg / mL Aβ antibody overnight at room temperature and washed three times with PBST. Then, biotin-labeled Aβ... 1-40 The monomer diluent (AnaSpec, AS-61483-01, 4-fold serial dilution in the 0-1000 nM range) was applied to the plate and captured by the immobilized antibody. The plate was incubated at room temperature for 1 hour and washed three times with PBST. The plate was then incubated at room temperature for 20 minutes with HRP-conjugated streptavidin (Thermo Fisher Scientific, SNN2004) diluted 1:5000 in 1% BSA and washed three times. A TMB-stabilized chromogen substrate solution (Lifetech, SB02) was added to the plate, and the reaction was terminated by adding 1M sulfuric acid. The absorbance was measured at 450 nm using a microplate reader. Figure 4 This demonstrates the interaction between rfmAb-1 and Aβ across the entire measured concentration range. 1-40 The monomer exhibits weak binding, while rfmAb-2 shows a concentration-dependent binding to Aβ. 1-40 The strong binding to the monomers is consistent with the known affinity of each reference antibody for the Aβ monomers. These results indicate that capture ELISA can determine the affinity of anti-Aβ antibodies for Aβ monomers.

[0092] Antibody-dependent cell-mediated phagocytosis (ADCP) assay

[0093] The preparation of Aβ fibrils labeled with the HiLyte488 fluorophore (AnaSpec, AS-60479-01; anaspec.com) was described below. First, 0.1 mg of HiLyte488-labeled Aβ fibrils were resuspended in 18 μL of 12 mM NaOH. 1-42 Monomer and 0.5 mg of unlabeled Aβ resuspended in 90 μL of 12 mM NaOH 1-42The monomer (rPeptide, A-1167-1) was mixed. Next, 680 μL of 10x PBS pH 7.4 (Gibco 70011-044) was added to both the labeled and unlabeled Aβ. 1-42 In a mixture of monomers, the mixture was incubated at 37°C for 2 hours to form HiLyte488-labeled Aβ. 1-42 The fibrils were then centrifuged at 16,000g for 5 minutes at 4°C to remove insoluble fibers. The supernatant was subjected to size exclusion chromatography (MSC). It was run in 200Increase 10 / 300GL (Cytiva28-9909-44) and separated from... Figure 1 The peaks in the original fiber are similar to the peaks in the original fiber.

[0094] Second, mouse microglia BV-2 cells (AccoGen, ABC-TC212S; accegen.com) were seeded at a density of 60,000 cells / well in 96-well cell culture plates and incubated overnight. Prior to ADCP assay, the cells were pretreated with ADCP assay diluent (containing DMEM / F12 and HEPES medium (Gibco, 11039021), 1% BSA, and 100 μg / mL fucoidan (Sigma-Aldrich, F8190)) at 37°C for 1 hour. 2.25 μg / mL HiLyte488 oligomer was mixed with serial dilutions (800 ng / mL, 160 ng / mL, 32 ng / mL, and 6.4 ng / mL) of Aβ antibody or mouse IgG2b (negative control) in the ADCP assay diluent for 30 minutes. The mixture was added to BV-2 cells and incubated at 37°C for 1.5 h to induce ADCP. The oligomer-antibody complex bound to the cell surface was removed by treatment with 0.25% trypsin at 4°C for 20 min. Cells were gently scraped from the culture plate and transferred to a cone-bottom 96-well plate (Thermo Scientific, 249935). Cells were then washed twice with ice-cold cell staining buffer (Baijin Biotechnology, 420201), fixed in ice-cold fixation buffer (Baijin Biotechnology, 420801) for 20 min, and washed once more. Finally, the fluorescence of the cells, reflecting the amount of HiLyte488-labeled oligomers acquired by the cells, was analyzed by flow cytometry.

[0095] Sequencing of Aβ fibrils binding monoclonal antibodies

[0096] The 33 Aβ fibrils-binding mAbs (Example 1) were tested using direct ELISA, competitive ELISA, and capture ELISA to assess their affinity for different Aβ conformations and their ability to mediate microglia uptake of Aβ fibrils in ADCP assays. The results are compiled in Table 1, where EC50 is based on the capture ELISA results. 50 IC in monomeric and competitive ELISA 50 The ratio of fibrils was used to rank the mAbs. This ratio reflects their selectivity for fibrils over monomers. Based on all data, mAbs 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A were selected as leader candidates. Of these leader candidates, mAbs 18P01A, 17P04A, and 20O07A were selected as the top three antibodies based on their high Aβ fibril selectivity; mAb 22D04A was selected due to its highest ADCP activity among the candidates; and 06E17A was selected due to its highest affinity for fibrils.

[0097] Table 1: Characterization and ranking of 33 anti-amyloid β monoclonal antibodies. (Calculated as EC50) 50 Monomer (capture ELISA) and IC 50 The fibrils (competitive ELISA) ratios are categorized in the list based on their selectivity for monomers. rfmAb-1 is the reference antibody for this study. The five antibodies in bold (18P01A, 17P04A, 20O07A, 22D04A, and 06E17A) were identified as lead candidates for further testing and development. UND, Undetermined value.

[0098]

[0099]

[0100] Example 3: Heavy and light chain variable region sequences of 15 anti-amyloid β monoclonal antibodies

[0101] Sequences of the variable regions of the fifteen (15) anti-Aβ mAbs of interest from Table 1 were obtained, and CDRs were identified using IMGT numbers (imgt.org / ). Table 2 below provides the amino acid sequences of the heavy chain (HC) variable region (VH) of each of the 15 anti-Aβ mAbs of interest from Table 1. Table 3 provides the amino acid sequences of the light chain (LC) variable region (VL) of each of the 15 anti-Aβ mAbs of interest from Table 1.

[0102] Table 2: Sequences of the HC variable region (VH) of the 15 mAbs of interest. In the complete VH amino acid sequence, CDR1-3 of each HC variable region are underlined, and CDRs are identified using IMGT numbers. Each CDR is also listed separately and identified by the SEQ ID NO: assigned to it.

[0103]

[0104]

[0105]

[0106] Table 3: Sequences of the LC variable region (VL) of 15 mAbs. In the complete VL amino acid sequence, CDR1-3 of each LC variable region are underlined, and CDRs are identified using IMGT numbers. Each CDR is also listed separately and identified by the SEQ ID NO: assigned to it.

[0107]

[0108]

[0109]

[0110] Example 4: Characterization of the leader antibody

[0111] Detailed profiles of five leading mAbs, 18P01A, 17P04A, 20O07A, 22D04A and 06E17A, were developed and are described below.

[0112] Affinity for Aβ peptide in direct ELISA

[0113] Each antibody was tested for Aβ using a direct ELISA. 1-42 and pyroglutamic acid-modified Aβ (Aβ) p3-42 The affinity of ) to the plate. The plate was treated with 0.5 μM Aβ. 1-42 (rPeptide, A-1167-2) or Aβ p3-42 (AnaSpec, AS-29907-01) The antibody was coated at 4°C for 2 hours. At room temperature, antibody diluent (3-fold serial dilutions in the 0-100 nM range) was added to the plate and allowed to stand for 1 hour. As described in Example 2, antibody binding to immobilized Aβ on each plate was detected using HRP-labeled secondary antibody and TMB substrate. Although all five leader antibodies showed binding to Aβ... 1-42 The strong binding, in which the calculated EC 50 <0.1nM( Figure 5A(and Table 1), but shows a correlation with Aβ p3-42 The weak binding, where the calculated EC 50 >50nM ( Figure 5B (and Table 1).

[0114] Selectivity for different Aβ conformations in competitive ELISA

[0115] Competitive ELISA was used to assess the relative affinity of the antibody for Aβ monomers, Aβ fibrils, and Aβ filaments. The antibody was subjected to competition from monomers, fibrils, or filaments on a monomer-coated plate. The antibody was pre-incubated for 1 hour at 4°C with a diluent for Aβ monomers, Aβ fibrils, or Aβ filaments (a 3-fold serial dilution starting at 3000 nM). The concentration of Aβ antibody used in the antibody-Aβ mixture was the EC50 of the antibody containing Aβ monomers in a direct ELISA. 90 The antibody Aβ mixture was then plated onto an ELISA plate coated with 0.5 μM Aβ monomer at room temperature for 10 minutes. Binding was detected using HRP-labeled secondary antibody and TMB substrate, as described in Example 2. In the competitive ELISA, all five leader antibodies showed high affinity for fibrils and low affinity for monomers (Figure 6 and Table 1). For all five antibodies, IC50... 50 Monomers and ICs 50 The ratio of fibrils was >300-fold, and for 18PO1 and 17PO4, the ratio was >1000-fold. The five antibodies showed higher affinity for fibrils than for monomers but lower affinity for fibrils. Due to the low affinity of the antibodies for monomers, the IC50 in competitive ELISA was low. 50 The monomer value is not reliable. Five antibodies were further tested in a capture ELISA to obtain their accurate affinity for the monomer.

[0116] Affinity for capturing Aβ monomers in ELISA

[0117] Capture ELISA can accurately measure the affinity of an antibody for an Aβ monomer. A 96-well plate was coated with 5 μg / mL antibody and incubated overnight at room temperature. The plate was then coated with biotin-labeled Aβ. 1-40 The monomer diluent (AnaSpec, AS-61483-01, 4-fold serial dilutions in the concentration range of 0-1000 nM) was used to treat the cells at room temperature for 1 hour. Biotin-labeled Aβ was detected by HRP-conjugated streptavidin and TMB as described in Example 2. 1-40 The binding of monomers to immobilized antibodies. This assay determines the antibody's binding to the Aβ monomer (EC). 50 EC (monomer) 50 ( Figure 7 (and Table 1). Then calculate the EC in the captured ELISA.50 IC in monomeric and competitive ELISA 50 The ratio of oligomers. This ratio reflects the antibody's selectivity for Aβ fibrils over Aβ monomers and is used to rank the antibodies as shown in Table 1.

[0118] Antibody-dependent cell-mediated phagocytosis (ADCP)

[0119] Based on the non-restrictive hypothesis that anti-Aβ antibodies can clear toxic Aβ fibrils / plaques in the brain by inducing ADCP in microglia, the ability of leader antibodies to mediate the phagocytosis of Aβ fibrils in microglia was assessed using HiLyte488-labeled Aβ fibrils and the mouse microglia cell line BV-2. HiLyte488-labeled Aβ... 1-42 Monomers and unlabeled Aβ 1-42 Monomers were mixed at a 1:5 ratio and incubated at 37°C for 2 hours to form HiLyte488-labeled fibrils, which were then purified by SEC separation and collection of fibril peaks. BV-2 cells were seeded at a density of 60,000 cells / well in 96-well cell culture plates and incubated overnight. Then, prior to ADCP assay, BV-2 cells were pretreated for 1 hour with ADCP assay diluent (DMEM / F12 and HEPES medium, 1% BSA, and 100 μg / mL fucoidan). Next, 2.25 μg / mL HiLyte488-labeled Aβ oligomers were mixed with sequential dilutions (800 ng / mL, 160 ng / mL, 32 ng / mL, and 6.4 ng / mL) of each leader antibody or mouse IgG2b (negative control) in the ADCP assay diluent for 30 minutes. A mixture of HiLyte488-labeled Aβ fibrils and antibody was added to BV-2 cells and incubated at 37°C for 1.5 h to induce ADCP. Oligomeric antibody complexes bound to the cell surface were removed by treatment with 0.25% trypsin at 4°C for 20 min. Cells were transferred to conical-bottom 96-well plates, washed twice with ice-cold cell staining buffer (Baijin Biotechnology Co., Ltd. #420201), fixed in ice-cold fixation buffer (Baijin Biotechnology Co., Ltd. #420801) for 20 min, and washed once more. Finally, the positive FITC signal of the cells was analyzed by flow cytometry, which proportionally reflected the amount of HiLyte488-labeled Aβ fibrils absorbed by BV-2 cells. Figure 8The raw RFU (relative fluorescence units) values ​​were used to report ADCP responses (Table 1), and the ability of each leader antibody candidate to trigger ADCP was assessed relative to the ADCP response of rfmAb-1. The relative ADCP values ​​of antibodies 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A were 1.36, 0.79, 1.05, 1.39, and 0.95, respectively.

[0120] Example 5: Reformatting and Reorganizing Expressions

[0121] Reformat to fully human antibody

[0122] Initially, as described in Example 1, five leader antibodies, 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A, were isolated from the hybridoma supernatant. They were then subcloned and purified to produce chimeric monoclonal antibodies with human F(ab')2 and mouse IgG1 CH2 and CH3 domains (i.e., chimeric human F(ab')2 / mouse IgG1 antibodies as described above). As shown in Tables 2 and 3: 18P01A has the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:61 and the light chain variable region (VL) amino acid sequence of SEQ ID NO:66; 17P04A has the VH amino acid sequence of SEQ ID NO:41 and the VL amino acid sequence of SEQ ID NO:46; 20O07A has the VH amino acid sequence of SEQ ID NO:71 and the VL amino acid sequence of SEQ ID NO:76; 22D04A has the VH amino acid sequence of SEQ ID NO:131 and the VL amino acid sequence of SEQ ID NO:136; and 06E17A has the VH amino acid sequence of SEQ ID NO:1 and the VL amino acid sequence of SEQ ID NO:6. The original chimeric leader antibodies can be identified as 18P01A (mIgG1), 17P04A (mIgG1), 20O07A (mIgG1), 22D04A (mIgG1) and 06E17A (mIgG1) as shown in Tables 4 and 5.

[0123] Then, through de novo gene synthesis, each of the five original chimeric human F(ab')2 / mouse IgG1 leader antibodies was reformatted to fully human IgG1λ by replacing the heavy chain constant region of each antibody with the human IgG1 heavy chain constant region, including CH1, the hinge region, CH2, and CH3. Therefore, the heavy chain (HC) amino acid sequence of each reformatted fully human antibody includes the VH amino acid sequence of the source mAb (including HC CDR1-3), and the light chain (LC) amino acid sequence of each reformatted fully human antibody includes the VL amino acid sequence of the source mAb (including LC CDR1-3).

[0124] The fully human antibody 18P01A (hIgG1) has the HC amino acid sequence of SEQ ID NO:157, including the 18P01A VH amino acid sequence of SEQ ID NO:61; and the LC amino acid sequence of SEQ ID NO:158, including the 18P01AVL amino acid sequence of SEQ ID NO:66. The fully human antibody 17P04A (hIgG1) has the HC amino acid sequence of SEQ ID NO:153, including the 17P04A VH amino acid sequence of SEQ ID NO:41; and the LC amino acid sequence of SEQ ID NO:154, including the 17P04A VL amino acid sequence of SEQ ID NO:46. The fully human antibody 20O07A (hIgG1) has the HC amino acid sequence of SEQ ID NO:161, including the 20O07A VH amino acid sequence of SEQ ID NO:71; and the LC amino acid sequence of SEQ ID NO:162, including the 20O07AVL amino acid sequence of SEQ ID NO:76. The fully human antibody 22D04A (hIgG1) has the HC amino acid sequence of SEQ ID NO:165, including the 22D04AVH amino acid sequence of SEQ ID NO:131; and the LC amino acid sequence of SEQ ID NO:166, including the 22D04AVL amino acid sequence of SEQ ID NO:136. The fully human antibody 06E17A (hIgG1) has the HC amino acid sequence of SEQ ID NO:169, including the 06E17AVH amino acid sequence of SEQ ID NO:1; and the LC amino acid sequence of SEQ ID NO:169, including the 06E17A VL amino acid sequence of SEQ ID NO:6.

[0125] The reformulated fully human antibody was expressed in CHO cells via transient transfection using the Expi CHO expression system and purified using a Protein A column. The purified fully human antibody was then tested in a series of in vitro assays, including direct ELISA, competitive ELISA, and capture ELISA, and the results were compared with the original chimeric antibody containing human F(ab')2 / mouse IgG1CH2-CH3. The results are presented in Table 4.

[0126] For the 17P04A, 20O07A, 22D04A, and 06E17A antibodies, their profiles showed no difference between the fully human version and the original chimeric human F(ab')2 / mouse IgG1 version. However, the fully human 18P01A showed a significantly reduced affinity for Aβ in all three conformations as tested by competitive ELISA. Results from direct ELISA showed that the EC50 of the fully human 18P01A was significantly lower than that of the original chimeric 18P01A at 0.062 mM.50 Aβ 1-42 It increased by more than 800 times, reaching a value of 49.96nM.

[0127] Table 4: Comparison of anti-Aβ antibodies in the fully human version and the mouse IgG1 / human chimeric version. UND, undetermined value.

[0128]

[0129] Reformatted human F(ab')2 / mouse IgG2a CH2-CH3 chimeric antibody

[0130] Converting mAbs from mouse IgG1 to mouse IgG2a enhances their ADCP activity. To test their effect on ADCP, the original leader antibodies 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A were reformatted from the original mouse IgG1 / human chimeric version (mouse IgG1 / human F(ab')2) to the mouse IgG2a / human chimeric version (mouse IgG2a CH2-CH3 / human F(ab')2) for animal studies. The reformatted antibody 18P01A (mIgG2a) has the heavy chain (HC) amino acid sequence of SEQ ID NO:155, including the 18P01AVH amino acid sequence of SEQ ID NO:61; and the LC amino acid sequence of SEQ ID NO:156, including the 18P01AVL amino acid sequence of SEQ ID NO:66. The reformatted antibody 17P04A (mIgG2a) has the HC amino acid sequence of SEQ ID NO:151, including the 17P04A VH amino acid sequence of SEQ ID NO:41; and the LC amino acid sequence of SEQ ID NO:152, including the 17P04A VL amino acid sequence of SEQ ID NO:46. The reformatted antibody 20O07A (mIgG2a) has the HC amino acid sequence of SEQ ID NO:159, including the 20O07A VH amino acid sequence of SEQ ID NO:71; and the LC amino acid sequence of SEQ ID NO:160, including the 20O07A VL amino acid sequence of SEQ ID NO:76. The reformatted antibody 22D04A (mIgG2a) has the HC amino acid sequence of SEQ ID NO:163, including the 22D04A VH amino acid sequence of SEQ ID NO:131; and the LC amino acid sequence of SEQ ID NO:164, including the 22D04AVL amino acid sequence of SEQ ID NO:136. The reformulated antibody 06E17A (mIgG2a) has the HC amino acid sequence of SEQ ID NO:167, including the 06E17A VH amino acid sequence of SEQ ID NO:1; and the LC amino acid sequence of SEQ ID NO:168, including the 06E17A VL amino acid sequence of SEQ ID NO:6.

[0131] Reformatted antibodies 17P04A (mIgG2a) and 18P01A (mIgG2a) were recombinantly expressed and purified on a large scale. Antibodies 17P04A (mIgG2a) and 18P01A (mIgG2a) were then tested in a series of assays including direct ELISA, competitive ELISA, capture ELISA, and ADCP. No significant differences were observed between the human F(ab')2 / mouse IgG2a CH2-CH3 chimeric form and the original human F(ab')2 / mouse IgG1 chimeric antibody in terms of selectivity for different Aβ conformations and ability to induce ADCP (Table 5).

[0132] Table 5: Comparison of 17P04A and 18P01A in the original human F(ab')2 / mouse IgG1 chimeric version and the reformulated human F(ab')2 / mouse IgG2a CH2-CH3 version. UND, values ​​not determined.

[0133]

[0134] Example 6: Pharmacokinetics of 17P04A and 18P01A in mice

[0135] Based on their high-level preferential binding to Aβ fibrils and their ability to trigger ADCP, 17P04A and 18P01A from the human F(ab')2 / mouse IgG2a CH2-CH3 chimeric version were selected as 17P04A (mIgG2a) and 18P01A (mIgG2a) for pharmacokinetic studies. Antibodies 17P04A (mIgG2a) and 18P01A (mIgG2a) were administered intraperitoneally (ip) to B6SJLF1 mice (The Jackson Laboratory, JAX#100012, jax.org; MPDID:178, phenomenon.jax.org / strains / 178) at a dose of 10 mg / kg. As a control, rfmAb-1, as rfmAB01 (mIgG2b), was also administered intraperitoneally at the same dose to a group of B6SJLF1 mice (The Jackson Laboratory, JAX#100012). Serum and brain tissue were collected at 4 hours, 8 hours, 1 day, 2 days, 3 days, 7 days, 10 days, and 14 days post-injection. Whole animals were perfused with PBS prior to brain tissue collection. Brain tissue was homogenized in 3 volumes (300 μL / 100 mg tissue) of TBS with a protease inhibitor mixture (Thermo Fisher Scientific, 78429; thermofisher.com) using a Bullet mixer 5E Gold (Next Advance, BB5E-AU) and zirconia beads (Next Advance, PINK5E100). The homogenate was transferred to 2 mL tubes and centrifuged at 16,000 x g for 20 min at 4 °C to remove precipitate. Whole blood was allowed to coagulate at room temperature for 30 min, then centrifuged and the separated serum was collected. The serum was then analyzed by using Aβ... 1-42 Direct ELISA using peptides as coating antigens measures drug concentrations in brain homogenates and serum supernatants. The plates were then coated with 0.5 μM Aβ. 1-42 (rPeptide, A-1167-2; rpeptide.com) The mixture was coated overnight at 4°C, washed three times with TBST, and blocked for 1 hour at room temperature with protein-free blocking buffer (Thermo Fisher 37572). Brain homogenate (initial dilution 1:4, 2-fold serial dilution, triplicate per concentration) or serum (initial dilution 1:200, 2-fold serial dilution, triplicate per concentration) was plated at room temperature for 1 hour. The antibodies in the brain homogenate or serum were detected by HRP-labeled secondary antibody and TMB substrate in relation to immobilized Aβ as described in the direct ELISA protocol in Example 2. 1-42The combination of [the two compounds] was performed. Non-compartmental pharmacokinetic (PK) analysis was conducted using an online PK calculator (available at dash.gallery / dash-pk-calc / ). The serum half-lives of rfmAb-1 (mIgG2a), 17P04A (mIgG2a), and 18P01A (mIgG2a) were 140.9 hours, 199.7 hours, and 183.2 hours, respectively. Figure 9 The half-lives of rfmAb-1 (mIgG2a), 17P04A (mIgG2a), and 18P01A (mIgG2a) in the brain were 28.2 hours, 92.4 hours, and 77.2 hours, respectively. Figure 10 Brain permeability (calculated as the ratio of AUC (area under the curve) in the brain to AUC in serum) was 1.9% for rfmAb-1 (mIgG2a), 5.5% for 17PO4A (mIgG2a), and 0.3% for 18PO1A (mIgG2a). Figure 10 In contrast, 17P04A (mIgG2a) has a longer half-life and better brain penetration in serum and brain than 18P01A (mIgG2a) or rfmAb-1 (mIgG2a). Despite the long half-life of 18P01A (mIgG2a) in serum and brain, its lower brain penetration should be considered as potentially limiting its efficacy in clearing Aβ in the brains of AD patients and in AD mouse models.

[0136] Example 7: Long-term efficacy study of 17P04A in 5xFAD mice

[0137] 5xFAD transgenic mice overexpress both mutant human amyloid-β precursor protein (APP) with mutations in Swedish (K670N, M671L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) and human presenile protein 1 (PSEN1) with two FAD mutations (M146L and L286V). These mice develop many AD-related phenotypes at a relatively early age. Here, 5xFAD mice are used as an AD mouse model to evaluate and compare the long-term efficacy of an antibody with the 17P04A variable region (17P04AFab), which exhibits high selectivity for Aβ oligomers / fibrils (see above) and rfmAb-1. Seven-month-old 5xFAD mice in a B6SJLF1 / J background (Jackson Lab, JAX#034840; jax.org; RRID: MMRRC_034840-JAX) were administered intraperitoneally weekly with 1 mg / kg, 3 mg / kg, and 10 mg / kg 17P04A (mIgG2a) or 1 mg / kg, 3 mg / kg, and 10 mg / kg rfmAb-1 (mIgG2a) for 16 weeks. 5xFAD mice administered weekly via intraperitoneal injection of 1x PBS (mediator) and age-matched naïve B6SJLF1 / J mice (wild-type) served as controls. Each treatment group contained 10–15 mice.

[0138] Aβ in fractions of brain homogenate 1-42 Level, Aβ 1-40 Measurement of levels and Aβ oligomer / fibril levels

[0139] Three (3) days after the last injection, mice were anesthetized with 250 mg / kg tribromoethanol and perfused with approximately 15 mL of 1x PBS (pH 7.4). Using a Bullet mixer (Next Advance, BB5E-AU), the hemicerebellum of each PBS-perfused mouse was homogenized in 600 μL of 1x Tris-buffered saline (TBS) (pH 7.4) with a mixture of protease and phosphatase inhibitors (Thermo Scientific, 78444). 250 μL of homogenate was centrifuged at 20,000 x g for 20 min at 4 °C. The supernatant was the TBS-soluble fraction. An equal volume of RIPA buffer (Thermo Scientific, 89901) was added to the remaining 250 μL of homogenate and centrifuged at 20,000 x g for 20 min at 4 °C. The supernatant was the RIPA-soluble fraction. The precipitate was resuspended in 100 μL of 8M guanidine hydrochloride and incubated at room temperature for 2 hours. The resuspension was diluted by adding 400 μL of 1x TBS and centrifuged again at 20,000 x g for 20 minutes at 4 °C. The supernatant was the guanidine-soluble fraction.

[0140] Following the manufacturer's instructions, Aβ in the RIPA soluble fraction and guanidine soluble fraction was measured using the Human Amyloid β (aa1-42) Quantikine ELISA Kit (R&D Systems, DAB142; rndsystems.com). 1-42 level. Figure 11A and 11B The results are shown in the image. Figure 11A As shown, Aβ in the RIPA soluble fraction (mainly Aβ monomer) 1-42 The level is not affected by any dose of 17P04A (mIgG2a) or rfmAb-1. Figure 11B The results showed that, when 17PO4A (mIgG2a) was injected at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, and mAb158 was injected at doses of 3 mg / kg and 10 mg / kg, the Aβ content in the guanidine soluble fraction (a fraction mainly containing insoluble Aβ filaments and plaques) was significantly reduced. 1-42 The level has decreased significantly. Importantly, Figure 11B The results showed that Aβ in mice treated with 17PO4 (mIgG2a) was... 1-42 The levels were significantly lower than those in mice treated with the same dose of rfmAb-1 (3 mg / kg rfmAb-1 vs. 3 mg / kg 17P04A(mIgG2a), p<0.05; 10 mg / kg rfmAb-1 vs. 10 mg / kg 17P04A(mIgG2a), p<0.01), indicating that 17P04A(mIgG2a) is more effective at removing Aβ plaques than the reference antibody rfmAb-1.

[0141] Aβ 1-40 It is an abundant Aβ isoform in the brain. It is considered non-toxic because it does not readily aggregate. Aβ in the RIPA soluble fraction and guanidine soluble fraction was measured using the Human Amyloid β (aa1-40) Quantikine ELISA Kit (R&D Systems, DAB140; rndsystems.com) according to the manufacturer's instructions. 1-40 level. Figure 12A and 12B The results are shown in the figure. RIPA soluble fraction for each sample ( Figure 12A ) and guanidine soluble fraction ( Figure 12B Aβ in both 1-40Levels were not affected by weekly intraperitoneal injections of 17PO4 (mIgG2a) at doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg, or by rfmAb-1 at doses of 3 mg / kg or 10 mg / kg. For unknown reasons, treatment with 1 mg / kg rfmAb-1 significantly increased Aβ levels in the soluble fraction of RIPA. 1-40 level.

[0142] Using a sandwich ELISA with rfmAb-1 as the capture antibody and biotin-labeled 18P01 as the detection antibody, the levels of Aβ oligomers / fibrils in the TBS soluble fraction of the brain were measured. Figure 13 The results are shown in the figure. For the assay, a 96-well plate was coated overnight at 4°C with 100 μL / well of PBS containing 2 μg / mL rfmAb-1. The plate was then blocked with PBS containing 1% BSA at room temperature for 1 hour. Then, 100 μL / well of soluble fraction of TBS (diluted 1 / 40) or purified Aβ was added. 1-42 Oligomers (2-fold serial dilutions in the range of 0–2000 pg / mL) were added to the plates. The plates were incubated at room temperature for 1 hour and washed 3 times. The plates were then incubated at room temperature for 1 hour and washed 3 times with 100 μL / well of 1% BSA containing 1 μg / mL biotinylated 18PO1. The plates were then incubated at room temperature for 1 hour and washed 3 times with 100 μL / well of HRP-conjugated streptavidin (Molecular Probes, SNN2004) diluted 1:5000 in 1% BSA. A TMB-stabilized chromogen substrate solution was added to the plates, and the reaction was terminated by adding 1M sulfuric acid. The absorbance was measured at 450 nm using a microplate reader. Aβ oligomer levels in WT mice were considered background signals and were subtracted from all groups. The results showed that treatment with 17PO4 reduced Aβ oligomers in the brain in a dose-dependent manner (reducing by 5%, 34%, and 68%, respectively, at 1 mg / kg, 3 mg / kg, and 10 mg / kg). Figure 13 The levels of Aβ oligomers / fibrils achieved by treatment with 10 mg / kg 17P04A (mIgG2a) were significantly lower than those achieved by treatment with a mordant (Veh) (i.e., the control). Treatment with 10 mg / kg rfmAb-1 reduced Aβ oligomers by 36% compared to the mordant-treated group (control), but this difference was not statistically significant. These results indicate that 17P04A (mIgG2a) removes Aβ oligomers / fibrils from the brain. These results suggest that 17P04A (mIgG2a) is more effective than rfmAb-1 in removing Aβ oligomers / fibrils from the brain.

[0143] Aβ 1-40 and Aβ1-42 It exists primarily in serum in monomeric form. Following the kit instructions, LEGENDMAX was used. TM Human amyloid β (1-42) ELISA kit (Baijin Biotechnology Co., Ltd., 448707) measures Aβ in the serum of treated mice. 1-42 Levels (Figure 14). Serum Aβ levels in mice treated with 1 mg / kg or 3 mg / kg 17PO4A (mIgG2a) or 1 mg / kg, 3 mg / kg or 10 mg / kg rfmAb-1 1-42 The levels remained unchanged. For unknown reasons, serum Aβ levels in mice treated with 10 mg / kg 17P04A (mIgG2a) were... 1-42 The level increased significantly. Using LEGEND MAX TM Human amyloid β (1-40) ELISA kit (Baijin Biotechnology Co., Ltd., 449007) measures Aβ in the serum of treated mice. 1-40 Levels (Figure 14). Serum Aβ 1-40 The levels were not affected by any dose of rfmAb01 or 17P04A (mIgG2a) treatment.

[0144] Measurement of plaques in fixed brain tissue

[0145] The other half of the brain from each PBS-perfused mouse was fixed in PBS containing 4% PFA for 1 day and incubated in 1x PBS for 1 day before being sent to NeuroScience Associates (neuroscienceassociates.com) for Campbell-Switzer staining to reveal Aβ plaques. Twenty-five (25) brain hemispheres were embedded together in a single block and frozen sectioned at 35 μm in the coronal plane through the cortical portion of the mouse brain hemispheres. Campbell-Switzer staining was performed through the brain on every 6th segment spaced at 210 μm intervals. Slides (25 brain slices per slide) were washed three times in dH2O, placed in 2% NH4OH for 5 minutes, and washed once more in dH2O. The slides were then placed in silver-pyridine-carbonate solution (NeuroScience Associates) for 40 minutes and washed in 1% citric acid for 3 minutes. Slides were placed in 4.99 pH acetate buffer working solution (Neuroscience Association) and then developed in fresh physical contrast agent ABS solution (Neuroscience Association). Development time was consistent across slides. Development was stopped by briefly placing the slide in 4.99 pH acetate buffer working solution. Slides were washed in dH2O for 30 seconds and then placed in 0.5% sodium thiosulfate solution for 45 seconds. After a final wash in dH2O (3 x 2 min), the slides were covered with coverslips. The slides were then imaged under a bright-field microscope. Representative images of Campbell-Switzer staining are shown in Figure 15. The area percentage of condensed and diffuse plaques in each brain slice was quantified using ImageJ software (imagej.net) for image processing and analysis. The cortex and hippocampus in each brain slice were outlined, and the total area was measured. To measure condensed plaques, the image thresholds were adjusted to 0 and 20. To measure condensed and diffuse plaques, the image thresholds were adjusted to 0 and 50. The particle analysis function in ImageJ was used to measure plaque area. Particle size was set from 10 to infinity. Area percentage was calculated as plaque area divided by total area. The area percentage of four brain slices located at -0.25 mm, -1.30 mm, -2.35 mm, and -3.40 mm of the anterior fontanelle in each brain was measured and averaged. Eight to 13 brains from each treatment group were analyzed. The results in Figure 15 show that treatment with 3 mg / kg and 10 mg / kg 17P04A (mIgG2a) significantly reduced condensed and diffuse plaques in the brains of 5xFAD mice. rfmAb-1 treatment at any dose had no significant effect on the level of condensed or diffuse plaques.

[0146] Results of long-term efficacy studies showed that treatment with 17PO4A (mIgG2a) significantly reduced the levels of soluble Aβ oligomers / fibrils in the brains of 5xFAD mice compared to levels in control 5xFAD mice treated with the same amount of mordant (PBS). Figure 13 This indicates that 17P04A (mIgG2a) effectively removes soluble Aβ oligomers / fibrils from the brain in an in vivo AD disease model.

[0147] Results of long-term efficacy studies showed that treatment with 17P04A (mIgG2a) significantly reduced the amount of condensed and insoluble plaques in the brains of 5xFAD mice compared to the amount treated with the same amount of mordant (PBS) for the same duration (Figure 15). These results demonstrate that 17P04A (mIgG2a) is more effective at removing both condensed and diffuse plaques in the brains of 5xFAD mice than the reference antibody rfmAb-1. This indicates that 17P04A (mIgG2a) effectively removes both condensed and insoluble Aβ filaments / plaques in the brain in an in vivo AD disease model. These results demonstrate that treatment with 17P04A (mIgG2a) can effectively remove and / or reduce Aβ plaques in the brain, thereby reducing plaque burden, in animals suffering from at least one symptom associated with Aβ protein aggregation, particularly AD.

Claims

1. An anti-amyloid β (Aβ) fibrils / oligomery antibody, wherein the antibody preferentially binds to soluble Aβ fibrils / oligomery compared to binding to Aβ monomers, wherein a complex comprising the anti-Aβ fibrils / oligomery antibody bound to the Aβ fibrils / oligomery is capable of triggering antibody-dependent cell-mediated phagocytosis (ADCP) of microglia upon exposure to the complex, wherein the anti-Aβ fibrils / oligomery antibody has a fully human variable region and includes a fully human heavy chain variable region (VH), the fully human VH comprising a heavy chain (HC) complementarity-determining region (CDR) 1 consisting of the amino acid sequence GFTLSSFS (SEQ ID NO: 42), an HC CDR 2 consisting of the amino acid sequence ISSRRTYI (SEQ ID NO: 43), and an HC consisting of the amino acid sequence ARGGYIGSPNAYDI (SEQ ID NO: 44). CDR3; and a fully human light chain variable region (VL), the fully human VL comprising a light chain (LC) CDR1 composed of the amino acid sequence TGAVTSDYY (SEQ ID NO: 47), an LC CDR2 composed of the amino acid sequence SAS, and an LC CDR3 composed of the amino acid sequence LLYYGGAWV (SEQ ID NO: 49).

2. The anti-Aβ fibrils / oligomery antibody according to claim 1, wherein the anti-Aβ fibrils / oligomery antibody is capable of penetrating the brain tissue of a mammalian individual after administration to the individual.

3. The anti-Aβ fibrils / oligomery antibody according to claim 2, wherein the anti-Aβ fibrils / oligomery antibody can reduce the level of soluble Aβ oligomers / fibrils in the brain of the mammalian individual after administration.

4. The anti-Aβ fibrils / oligomery antibody according to claim 2, wherein the anti-Aβ fibrils / oligomery antibody can reduce the level of Aβ plaques in the brain of the mammalian individual after administration.

5. The anti-Aβ fibrils / oligomery antibody according to claim 1, wherein the constant region sequence involved in triggering ADCP includes a sequence from the crystallizable region (Fc) of the IgG fragment.

6. The anti-Aβ fibrils / oligomer antibody according to claim 5, wherein the Fc is selected from mouse IgG1 Fc, human IgG1 Fc and mouse IgG2a Fc.

7. The anti-Aβ fibrils / oligomery antibody according to claim 1, wherein the anti-Aβ fibrils / oligomery antibody comprises a fully human heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 41; and a fully human light chain variable region (VL) having the amino acid sequence of SEQ ID NO:

46.

8. A pharmaceutical composition comprising the anti-Aβ fibrils / oligomer antibody of claim 1 and a pharmaceutically acceptable carrier or excipient.

9. Use of the anti-Aβ fibrils / oligomer antibody of claim 1 in the preparation of a medicament for preventing or slowing the development of at least one symptom associated with Aβ protein aggregation in an individual in need, wherein the symptom associated with Aβ protein aggregation is Alzheimer's disease (AD).

10. The use according to claim 9, wherein the anti-Aβ fibrils / oligomery antibody reduces the amount of soluble Aβ fibrils / oligomers in the brain tissue of the individual.

11. The use according to claim 9, wherein the anti-Aβ fibrils / oligomery antibody reduces the amount of Aβ plaques in an individual.

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