Anti-amyloid beta protofibril / oligomer antibodies and uses thereof

Abstract

The present disclosure provides anti-amyloid beta (Aβ) antibodies and antibody fragments that preferentially bind soluble amyloid Aβ protofibrils / oligomers and initiate ADPC in microglial cells, anti-amyloid beta (Aβ) antibodies and antibody fragments that reduce the levels of soluble amyloid Aβ protofibrils / oligomers and insoluble amyloid Aβ plaques in brain tissue, and uses of anti-Aβ protofibril / oligomer antibodies and antibody fragments in the treatment, prevention, diagnosis, screening, and monitoring of conditions associated with Aβ protein aggregation, particularly Alzheimer's disease (AD).

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 440,345, filed January 20, 2023, entitled "Anti-Amyloid Beta Oligomer Antibodies and Uses Thereof," the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing, which has been electronically submitted in XML file format and is incorporated herein by reference in its entirety. This XML copy was created on January 17, 2024, is named 1121-103PCT_SL.xml, and is 187,258 bytes in size.

[0003] The present disclosure relates to antibodies and antibody fragments that preferentially bind soluble amyloid beta (Aβ) protofibrils / oligomers, and the use of anti-Aβ protofibril / oligomer antibodies and antibody fragments in the treatment, prevention, diagnosis, screening, and monitoring of conditions associated with Aβ protein aggregation, specifically Alzheimer's disease (AD). [Background technology]

[0004] Neurodegenerative conditions associated with the aggregation and deposition of amyloid beta (Aβ) protein include Alzheimer's disease (AD), a common neurodegenerative disorder. Patients with AD eventually lose the ability to perform daily activities due to memory loss, mood / behavioral changes, and other disability impairments. More than 6 million Americans live with AD in the United States, and this number is expected to reach 14 million by 2060. There are more than 55 million people with dementia worldwide, with AD contributing to 60–70% of cases. In 2019, the global economic burden of AD and related dementias was estimated at $2.8 trillion. This figure is expected to increase to $16.9 trillion by 2050. There are seven FDA-approved medications for AD, but none have demonstrated disease-modifying benefits.

[0005] AD is classified into familial AD (fAD) and late-onset AD (LOAD). Mutations in APP and PSEN1 have been reported to cause fAD. Both genes are responsible for the production of Aβ peptides, which are released from the amyloid beta precursor protein (APP) by proteolytic cleavage involving presenilin 1, encoded by the PSEN1 gene. The cause of LOAD is unknown. Amyloid plaques and tau tangles are the most prominent pathological features of the AD brain. Amyloid plaques are aggregated deposits of Aβ and generally form before the appearance of tau tangles. The onset of LOAD is highly correlated with the appearance of amyloid deposits, and the severity or stage of the disease correlates with the level of tau tangles. Therefore, the main efforts in AD treatment development focus on targeting Aβ deposits and tau tangles. Aβ may be a better therapeutic target for AD than tau because (1) Aβ resides in the extracellular space, potentially more accessible to biologics than tau tangles, and (2) Aβ pathology is upstream of tau pathology.

[0006] Aβ peptides exist in different forms, including monomers, aggregates containing soluble protofibrils / oligomers, and insoluble plaques. The soluble protofibril / oligomer forms are believed to be toxic to neurons, while the insoluble plaque forms, which result in the deposition of aggregated Aβ fibrils, are believed to be inactive. Aβ has been the primary therapeutic target for AD drug development for over 20 years. Both monomeric and aggregated forms of Aβ have been targeted. To date, monoclonal antibodies targeting Aβ monomers have failed to clear amyloid plaques or provide cognitive benefits. A total of five mAbs targeting extracellular aggregated Aβ (aducanumab, crenezumab, lecanemab, donanemab, and gantenerumab) have been tested in phase 2 or 3 clinical trials. All but one of these antibodies (crenezumab) were effective in clearing amyloid plaques in a dose-dependent manner in human patients (crenezumab was an IgG4 isotype that may not have strong effector function). Aducanumab and lecanemab were reported to slow cognitive decline in phase 3 trials. Donanemab was reported to slow cognitive decline in phase 2 trials. These data support the idea that monoclonal antibodies can reach their brain targets when used at high doses and that aggregated Aβ may be a reasonable target for AD. Lecanemab preferentially binds Aβ protofibrils / oligomers and causes a lower incidence of edema than aducanumab and gantenerumab, which preferentially bind Aβ plaques. Summary of the Invention

[0007] The present disclosure provides anti-Aβ protofibril / oligomer antibodies and antibody fragments that preferentially bind Aβ protofibrils / oligomers, amino acid sequences of the variable regions of anti-Aβ protofibril / oligomer antibodies and antibody fragments, nucleotide sequences encoding the variable regions of anti-Aβ protofibril / oligomer antibodies and antibody fragments, and methods for identifying, producing, and using anti-Aβ protofibril / oligomer antibodies and antibody fragments. The anti-Aβ protofibril / oligomer antibodies and antibody fragments provided herein have fully human variable regions comprising fully human heavy chain variable region (VH) sequences and fully human light chain variable region (VL) sequences. The disclosure further provides fully human anti-Aβ protofibril / oligomer antibodies and antibody fragments that preferentially bind Aβ protofibrils / oligomers, comprising fully human heavy chain variable region and fully human light chain variable region sequences.

[0008] The present disclosure provides anti-Aβ protofibril / oligomer antibodies and antibody fragments thereof that preferentially bind to soluble Aβ protofibrils / oligomers, and the anti-Aβ protofibril / oligomer antibodies and antibody fragments thereof can induce antibody-dependent cell-mediated phagocytosis (ADCP) in cells, including microglial cells, exposed to the anti-Aβ protofibril / oligomer antibodies and antibody fragments bound to Aβ protofibrils / oligomers. Also provided are anti-Aβ protofibril / oligomer antibodies and antibody fragments thereof that can penetrate brain tissue after administration to a mammalian subject. Also provided are anti-Aβ protofibril / oligomer antibodies and antibody fragments thereof that can reduce soluble Aβ oligomers / protofibrils in brain tissue after administration to a mammalian subject. Provided are anti-Aβ protofibril / oligomer antibodies and antibody fragments thereof that can reduce insoluble Aβ fibrils / plaques, including both dense and diffuse plaques, in brain tissue after administration to a mammalian subject. The anti-Aβ protofibril / oligomer antibodies and antibody fragments having fully human variable regions provided herein have a low risk of adverse reactions or undesirable side effects caused by immunogenicity when administered to a mammalian subject, particularly a human subject.

[0009] The present disclosure provides anti-Aβ protofibril / oligomer antibodies and antibody fragments for use in methods for treating, preventing, preventing, diagnosing, screening, and monitoring conditions associated with aggregation of Aβ protein, including, but not limited to, Alzheimer's disease (AD).

[0010] The present disclosure provides 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 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:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ :43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, 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:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:76, 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 NO:86 , 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 ID NO: 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 ID NO: 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 ID NO: 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 ID NO: 147, and SEQ ID NO: 149.

[0011] The present disclosure provides antibodies and antibody fragments comprising a 06E17A 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 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; and antibodies and antibody fragments comprising a 17D08A 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: 26. antibodies and antibody fragments comprising the 17H05A variable region, which comprises 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; antibodies and antibody fragments comprising the 17P04A variable region, which comprises 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; antibodies and antibody fragments comprising the 18F06A variable region, which comprises 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 and antibody fragments; antibodies and antibody fragments comprising an 18P01A variable region comprising a fully human VH having the amino acid sequence of SEQ ID NO: 61 and a fully human VL having the amino acid sequence of SEQ ID NO: 66; antibodies and antibody fragments comprising a 20O07A variable region comprising 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; antibodies and antibody fragments comprising a 20O11A variable region comprising 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. fragments; antibodies and antibody fragments comprising a 21F12A variable region comprising 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 comprising 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 comprising 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;Anti-Aβ protofibril / oligomer antibodies and antibody fragments are provided, which are identified as antibodies and antibody fragments comprising a 21P22A variable region comprising a fully human VH having the amino acid sequence of SEQ ID NO: 121 and a fully human VL having the amino acid sequence of SEQ ID NO: 126; antibodies and antibody fragments comprising a 22D04A variable region comprising 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; antibodies and antibody fragments comprising a 22H10A variable region comprising 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 anti-Aβ protofibril / oligomer antibodies and antibody fragments having at least 85% amino acid sequence identity to the VH amino acid sequences and / or VL amino acid sequences described above.

[0012] The present disclosure provides an antibody or antibody fragment comprising: (a) a heavy chain variable region (VH) comprising an HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO: 42), an HC CDR2 having the amino acid sequence ISSRRTYI (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) comprising 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) a VH comprising 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 TSHAPNFDAFDI (SEQ ID NO: 64), an LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 67), an LC CDR2 having the amino acid sequence DNS (c) an antibody or antibody fragment comprising a VH comprising an HC CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO: 72), an HC CDR2 having the amino acid sequence IRSKVNSYAT (SEQ ID NO: 73), and an HC CDR3 having the amino acid sequence TSHAPIFDAFDI (SEQ ID NO: 74), and a VL comprising an LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 77), an LC CDR2 having the amino acid sequence DNS, and an LC CDR3 having the amino acid sequence GTWDSSLSTYF (SEQ ID NO: 79); (d) an antibody or antibody fragment comprising a VH comprising an HC CDR1 having the amino acid sequence GFTFSNAW (SEQ ID NO: 132), an HC CDR2 having the amino acid sequence IKSKTDGGTR (SEQ ID NO: 133), and an HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 134), and an LC CDR3 having the amino acid sequence SSNIKSNT (SEQ ID NO: 137). an antibody or antibody fragment comprising a VL comprising a CDR1, a LC CDR2 having the amino acid sequence RNN, and a LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 139);and (e) an antibody or antibody fragment comprising a VH comprising an HC CDR1 having the amino acid sequence GFSFSNAW (SEQ ID NO: 2), an HC CDR2 having the amino acid sequence IKSKTDGGTI (SEQ ID NO: 3), and an HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 4), and a VL comprising an LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO: 7), an LC CDR2 having the amino acid sequence RNN, and an LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 9);

[0013] The present disclosure provides anti-Aβ protofibril / oligomer antibodies and antibody fragments having constant region sequences including a sufficient fragment crystallizable region (Fc) sequence to enable Fc-mediated effector functions such as antibody-dependent cellular phagocytosis (ADCP). The present disclosure provides anti-Aβ protofibril / oligomer antibodies and antibody fragments having Fc sequences derived from the IgG class of immunoglobulin molecules. The present disclosure provides anti-Aβ protofibril / oligomer antibodies and antibody fragments having constant region Fc sequences that can be mouse IgG1 Fc, human IgG1 Fc, mouse IgG2a Fc, and mouse IgG2b Fc.

[0014] The present disclosure provides pharmaceutical compositions containing at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0015] The present disclosure provides a method for reducing the amount of soluble Aβ protofibril / oligomers in a subject by administering to the subject a therapeutically effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. One method for reducing the amount of soluble Aβ protofibril / oligomers in a subject is provided, in which a therapeutically effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein is administered to the subject to elicit antibody-dependent cell-mediated phagocytosis (ADCP) of a complex comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to Aβ protofibril / oligomer. ADCP mediated by responsive effector cells can occur in any tissue, including at least one of blood, lymph, cerebrospinal fluid (CSF), neural tissue, and the brain.

[0016] The present disclosure provides methods for reducing the amount of soluble Aβ protofibrils / oligomers in the brain of a mammalian subject by administering to the subject a therapeutically effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein.

[0017] The present disclosure provides a method for reducing the amount of Aβ plaques in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. The present disclosure provides a method for reducing the level of Aβ plaques in the brain of a mammalian subject by administering to the subject a therapeutically effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. The present disclosure provides a method for reducing the level of dense Aβ plaques in the brain of a mammalian subject by administering to the subject a therapeutically effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. The present disclosure provides a method for reducing the level of diffuse Aβ plaques in the brain of a mammalian subject by administering to the subject a therapeutically effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. Thus, the present disclosure provides a method for reducing plaque burden in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. One method for reducing the amount of Aβ plaques in a subject is provided, in which administering to the subject a therapeutically effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein induces ADCP of a complex comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to Aβ protofibrils / oligomers, which is expected to result in a reduction in the amount of Aβ plaques in the subject.

[0018] The present disclosure provides methods for treating at least one condition associated with Aβ protein aggregation by administering an effective amount of at least one anti-Aβ oligomer antibody or antibody fragment disclosed herein to a subject in need thereof. One method for treating at least one condition associated with Aβ protein aggregation by administering an effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment thereof is provided, wherein the at least one condition associated with Aβ protein aggregation is Alzheimer's disease (AD).

[0019] The present disclosure provides a method for treating at least one condition associated with Aβ protein aggregation, in which at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein is administered to a subject in need thereof, thereby eliciting antibody-dependent cell-mediated phagocytosis (ADCP) of a complex comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to an Aβ protofibril / oligomer.

[0020] The present disclosure includes methods for preventing the onset or further development of at least one condition associated with Aβ protein aggregation by administering an effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein to a subject in need thereof. One method for preventing the onset or further development of at least one condition associated with Aβ protein aggregation is provided, in which administering an anti-Aβ protofibril / oligomer antibody or antibody fragment to a subject in need thereof elicits antibody-dependent cell-mediated phagocytosis (ADCP) of a complex comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to Aβ protofibril / oligomer, thereby substantially preventing Aβ protein aggregation and reducing plaque burden in the subject. A method is provided for treating or preventing the onset or further development of at least one condition associated with Aβ protein aggregation by administering an effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment, wherein the at least one condition associated with Aβ protein aggregation is Alzheimer's disease (AD).

[0021] The present disclosure provides methods for diagnosing at least one condition associated with aggregation of Aβ protein in a subject by detecting Aβ protofibrils / oligomers in a sample from the subject using at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein, wherein the sample can be a soluble fraction or a fixed tissue sample.

[0022] The present disclosure discloses a method of screening for at least one condition associated with aggregation of Aβ protein in a subject by detecting Aβ protofibrils / oligomers in a sample from the subject using an anti-Aβ protofibril / oligomer antibody or antibody fragment.

[0023] The present disclosure provides a method for monitoring at least one condition associated with Aβ protein aggregation in a subject, the method comprising detecting Aβ protofibrils / oligomers in a sample from the subject using at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein.

[0024] The present disclosure provides in vivo, ex vivo, or in vitro methods for detecting soluble Aβ protofibril / oligomers in a sample by contacting the sample with at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein and detecting binding of the anti-Aβ protofibril / oligomer antibody or antibody fragment to the Aβ protofibril / oligomer, indicating the presence of soluble Aβ protofibril / oligomers in the sample. The present disclosure provides in vivo, ex vivo, or in vitro methods for detecting soluble Aβ protofibrils / oligomers in a sample by contacting the sample with at least one disclosed anti-Aβ protofibril / oligomer antibody or antibody fragment and detecting binding of the anti-Aβ protofibril / oligomer antibody or antibody fragment to the Aβ protofibrils / oligomers, indicating the presence of soluble Aβ protofibrils / oligomers in the sample, wherein the Aβ peptide-binding polypeptide is part of a fusion protein or conjugate.

[0025] The present disclosure provides an in vivo, ex vivo, or in vitro method for reducing the amount of soluble Aβ protofibrils / oligomers in a sample suspected of containing Aβ protofibrils / oligomers, the method comprising contacting the sample with an effective amount of at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein to form a mixture, and then recovering the anti-Aβ protofibril / oligomer antibody or antibody fragment from the mixture, thereby removing Aβ protofibrils / oligomers bound to the anti-Aβ protofibril / oligomer antibody or antibody fragment from the sample.

[0026] The present disclosure provides methods for producing anti-Aβ protofibril / oligomer antibodies or antibody fragments that preferentially bind soluble Aβ protofibrils / oligomers and have a lower risk of immunogenicity, i.e., a lower risk of adverse reactions or other undesirable side effects, when administered to a subject, particularly a human. The disclosed methods include selecting a mammal capable of producing antibodies with fully human variable regions; immunizing the mammal by introducing a purified Aβ protofibril preparation into the mammal; harvesting enriched B cells from the spleen and lymph nodes of the mammal immunized with the purified Aβ protofibril preparation; fusing the B cells with a myeloma fusion partner to generate hybridomas; recovering antibodies from each hybridoma; and performing a primary screening of the recovered antibodies from each hybridoma to measure binding to Aβ peptide(s) in an ELISA-based assay and to determine whether the antibodies bind to the Aβ peptide(s). performing a primary screen, comprising identifying and recovering any antibodies with a detectable level of binding; performing a conformational screen of each recovered antibody after the primary screen, comprising measuring competition between Aβ monomers and Aβ protofibrils for binding to the recovered antibody using a competitive ELISA, and measuring the direct binding of each recovered antibody to Aβ monomers, Aβ protofibrils, and Aβ fibrils by ELISA, and further selecting each recovered antibody that exhibits a higher affinity for protofibrils than monomers in the competitive ELISA;

[0027] The present disclosure provides an isolated nucleic acid molecule encoding at least a portion of the anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein, wherein the nucleotide sequence of the isolated nucleic acid molecule is set forth in SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, 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:110, 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. The present disclosure provides a vector comprising an isolated nucleic acid molecule encoding at least a portion of an anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. [Brief explanation of the drawings]

[0028] Figure 1 shows the Aβ 1-42 Aβ by size exclusion chromatography (SEC) for the purpose of recovering purified preparations of protofibrils 1-42 Conformational analysis of Aβ is shown. 1-42 The monomer solution was incubated at 37°C for 2 hours to separate Aβ 1-42 A mixture of protofibrils and monomers was formed. 1-42 A control solution of monomer (dotted line) and a mixture of Aβ 1-42 protofibrils and monomer (solid black line) were analyzed by SEC on a Superdex® 200 Increase 10 / 300 GL column. 1-42 Monomer (dotted line) control sample and Aβ 1-42 The overlay of the SEC results for the mixture of protofibrils and monomers (black solid line) shows the Aβ 1-42 Protofibrils and Aβ 1-42 The area percentages are shown together with the area of ​​the monomer peak.1-42 The sample from the protofibril peak was used to immunize purified Aβ 1-42 They were collected as protofibrils.

[0029] Figures 2A-2B show the results obtained from direct ELISA of reference antibodies rfmAb-1 and rfmAb-2 for different Aβ conformations. 1-40 monomer), Aβ protofibril (Aβ 1-42 protofibrils), or Aβ fibrils (Aβ 1-42 rfmAb-1 and rfmAb-2 were applied to the plate, and antibody binding to Aβ in each well was detected with an HRP-conjugated secondary antibody and TMB substrate. Figure 2A: EC50 of rfmAb-1 against Aβ monomer (Aβ 1) measured by direct ELISA. 1-40 The EC50 of rfmAb-1 against Aβ protofibrils (Aβ 1-42 The EC50 of rfmAb-1 against Aβ and Aβ fibrils (Aβ protofibrils, open squares) was 0.04 nM. 1-42 The EC50 of rfmAb-2 against Aβ monomers (Aβ fibrils, open triangles) was 1.1 nM. Figure 2B: The EC50 of rfmAb-2 against Aβ monomers (Aβ fibrils, open triangles) was 1.1 nM. 1-40 The EC50 of rfmAb-2 against Aβ protofibrils (Aβ 1-42 protofibrils, open squares) was 0.05 nM, and the EC50 of rfmAb-2 against Aβ fibrils (Aβ 1-42 fibrils, open triangles) was 0.08 nM.

[0030] Figures 3A-3B show the results obtained from a competitive ELISA of the reference antibodies rfmAb-1 and rfmAb-2 for different Aβ conformations. Each antibody (rfmAb-1 at 0.12 nM and rfmAb-2 at 0.21 nM) was administered to the Aβ monomer (Aβ 1-40 monomer), Aβ protofibril (Aβ 1-42protofibrils), or Aβ fibrils (Aβ 1-42 The antibody-Aβ mixture was then applied to an ELISA plate coated with Aβ monomer for 10 min, and binding to Aβ monomer was detected using an HRP-conjugated secondary antibody and TMB substrate. Figure 3A: The calculated IC50 of Aβ monomer for rmAb-1 (Aβ fibrils) was calculated. 1-40 The IC50 of Aβ protofibrils against rmAb-1 (Aβ 1-42 protofibrils, open squares) was 1.0 nM, and the IC50 of Aβ fibrils against rfmAb-1 (Aβ 1-42 The IC50 of Aβ monomers against rfmAb-2 (Aβ fibrils, open triangles) was 128.7 nM. Figure 3B: The calculated IC50 of Aβ monomers against rfmAb-2 (Aβ 1-40 The IC50 of Aβ protofibrils against rfmAb-2 (Aβ 1-42 The IC50 of Aβ fibrils against rfmAb-2 (Aβ protofibrils, open squares) was 5.4 nM. 1-42 fibrils, open triangles) was 179.5 nM.

[0031] Figure 4 shows the results obtained from the capture ELISA of the reference antibodies rfmAb-1 and rfmAb-2. 96-well plates were coated with each antibody (100 ng per well). Biotin-conjugated Aβ 40 Dilutions of monomer were applied to the plate for 1 h, and binding was detected with HRP-conjugated streptavidin and TMB substrate. The EC50 for rfmAb-1 (filled circles) could not be reliably measured and was therefore considered unstable. The EC50 for rfmAb-2 (filled squares) was 0.067 nM.

[0032] Figures 5A-5B show the results of direct ELISA for the five lead antibodies 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A, and the reference antibody rfmAb-1. 1-42 (Figure 5A) or Aβ p3-42(Figure 5B) was coated onto the wells of an ELISA plate at 50 pmol per well. Dilutions of the antibody were then applied to the plate. Binding was detected with an HRP-labeled secondary antibody and TMB substrate. Figure 5A shows the Aβ 1-42 Direct antibody binding to protofibrils was demonstrated with EC50 values ​​of 0.062 nM, 0.045 nM, 0.050 nM, 0.035 nM, and 0.052 nM for 18P01A (open circles), 17P04A (open squares), 20O07A (upward-pointing triangles), 22D04A (downward-pointing triangles), and 06E17A (open diamonds), respectively. p3-42 The figure shows antibody binding to Aβ. Although EC50 values ​​could not be determined for all lead antibodies, this indicates that the binding of Aβ p3-42 This indicates weak or no binding to

[0033] Figures 6A-6E show the results of competitive ELISA for the five lead antibodies. Each antibody was tested against Aβ monomer (Aβ 1-40 monomer), Aβ protofibril (Aβ 1-42 protofibrils), or Aβ fibrils (Aβ 1-42 The antibody-Aβ mixture was then applied to an ELISA plate coated with Aβ monomer for 10 minutes, and binding was detected with an HRP-conjugated secondary antibody and TMB substrate. 1-40 monomers, open circles), Aβ protofibrils (Aβ 1-42 protofibrils (white squares), Aβ fibrils (Aβ 1-42 The results for each antibody against fibrils (open triangles) are shown in Figure 6A. Figure 6A shows the results for antibody 18P01A, Figure 6B shows the results for antibody 17P04A, Figure 6C shows the results for antibody 20O07A, Figure 6D shows the results for antibody 22D04A, and Figure 6E shows the results for antibody 06E17A.

[0034] Figure 7 shows the results obtained from the capture of soluble biotin-Aβ monomers using immobilized antibodies (0.5 μg per well) for each of the five lead antibodies: 18P01A (open circles), 17P04A (open squares), 20O07A (upward-pointing triangles), 22D04A (downward-pointing triangles), and 06E17A (open diamonds), as well as two reference antibodies: rfmAb-1 (closed circles) with low affinity for the monomer and rfmAb-2 (closed squares) with high affinity for the monomer.

[0035] Figure 8 shows the results of an ADCP assay using BV2 microglial cells for antibodies 18P01A (open circles), 17P04A (open squares), 22D04A (downward-pointing triangles), rfmAb-1 (closed circles), rfmAb-2 (closed squares), and mouse IgG2b (crosses) as a negative control. HiLyte488-labeled protofibrils (2.25 μg / mL) were mixed with a dilution series of antibodies (800, 160, 32, and 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. The positive fluorescent signal generated by the internalized HiLyte488-labeled protofibrils was analyzed by flow cytometry, and relative fluorescence values ​​were calculated.

[0036] Figure 9 shows serum PK data for antibody 17P04A (open squares), antibody 18P01A (open circles), and rfmAb-1 (filled circles) in the serum of B6SJLF1 mice. Values ​​for each time point were averaged from samples obtained from two to three animals. The table shows the calculated PK parameters for each antibody in serum.

[0037] Figure 10 shows the intracerebral PK data for antibody 17P04A (open squares), antibody 18P01A (open circles), and rfmAb-1 (filled circles) in the brains of B6SJLF1 mice. Values ​​for each time point were averaged from samples obtained from two to three animals. The table shows the calculated PK parameters for each antibody in serum.

[0038] Figures 11A-11B show Aβ levels in the RIPA-soluble (Figure 11A) and guanidine-soluble (Figure 11B) fractions of brain homogenates obtained from naive (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice treated with PBS vehicle by weekly IP injections (Veh), 11-month-old 5xFAD mice treated with rfmAb-1 by weekly IP injections of rfmAb-1 at 1, 3, or 10 mg / kg for 16 weeks, and 11-month-old 5xFAD mice treated with 17P04A by weekly IP injections of 1, 3, or 10 mg / kg for 16 weeks. N=13, 14, 8, 8, 10, 8, 9, 9. In Figure 11B, results of one-way ANOVA followed by Sidak's test: ** indicates p<0.01 for 3 or 10 mg / kg rfmAb-1 vs. PBS vehicle (Veh), **** indicates p<0.0001 for 1, 3, or 10 mg / kg 17P04A vs. PBS vehicle (Veh), # indicates p<0.05 for 3 mg / kg rfmAb-1 vs. 3 mg / kg rfmAb-1 and 17P04A, and ## indicates p<0.01 for 10 mg / kg rfmAb-1 vs. 10 mg / kg 17P04A.

[0039] Figures 12A-B show Aβ levels in the RIPA-soluble (Figure 12A) and guanidine-soluble (Figure 12B) fractions of brain homogenates obtained from naive (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice treated with PBS vehicle by weekly IP injections (Veh), 11-month-old 5xFAD mice treated with rfmAb-1 by weekly IP injections of rfmAb-1 at 1, 3, or 10 mg / kg for 16 weeks, and 11-month-old 5xFAD mice treated with 17P04A by weekly IP injections of 1, 3, or 10 mg / kg for 16 weeks. N=13, 14, 8, 8, 10, 8, 9, 9. In FIG. 12B, results of one-way ANOVA followed by Sidak's test: ** indicates p<0.01 for 1 mg / kg rfmAb-1 vs. PBS vehicle (Veh).

[0040] Figure 13 shows the levels of Aβ oligomers / protofibrils in the TBS-soluble fraction of brain homogenates from naive (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5×FAD mice (Veh) treated with PBS vehicle by weekly IP injections, 11-month-old 5×FAD mice treated with rfmAb-1 by weekly IP injections of 1, 3, or 10 mg / kg for 16 weeks, and 11-month-old 5×FAD mice treated with 17P04A by weekly IP injections of 1, 3, or 10 mg / kg for 16 weeks. Aβ oligomer levels were measured using a sandwich ELISA, where rfmAb-1 was used as the capture Ab and biotin-18P01A was used as the detection Ab. N=13, 14, 8, 8, 10, 8, 9, 9. Results of one-way ANOVA followed by Sidak test: *** indicates p<0.001 for 10 mg / kg 17P04 vs. vehicle (Veh).

[0041] Figures 14A-B show serum Aβ levels in naive (untreated) 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice (Veh) treated with weekly ip injections of PBS vehicle, 11-month-old 5xFAD mice treated with rfmAb-1 by weekly ip injections of rfmAb-1 at 1, 3, or 10 mg / kg for 16 weeks, and 11-month-old 5xFAD mice treated with 17P04A by weekly ip injections of 17P04A at 1, 3, or 10 mg / kg for 16 weeks. 1-42 (Figure 14A) and Aβ 1-40 The levels of 17P04 at 10 mg / kg and 17P04 at 20 mg / kg were measured (Figure 14A, N = 6-10), and those of 17P04 at 20 mg / kg and 17P04 at 20 mg / kg were measured (Figure 14B). Figure 14A shows N = 6-10, and Figure 14B shows N = 5-13. In Figure 14A, the results of one-way ANOVA followed by Sidak's test were shown: * indicates p<0.05 for 10 mg / kg 17P04 vs. vehicle (Veh).

[0042] Figures 15A-15B show Campbell-Switzer staining of brain sections from 11-month-old B6SJLF1 / J (WT) mice, 11-month-old 5xFAD mice (Veh) treated with PBS vehicle by weekly IP injections, 11-month-old 5xFAD mice treated with rfmAb-1 by weekly IP injections of rfmAb-1 at 1, 3, or 10 mg / kg for 4 months, and 11-month-old 5xFAD mice treated with 17P04A by weekly IP injections of 1, 3, or 10 mg / kg for 4 months (Figure 15A), as well as the estimated amount of diffuse and dense Aβ plaques in the brain (Figure 15B). Figure 15A is a representative image of Campbell-Switzer staining on brain sections at approximately bregma -1.30 mm. Figure 15B shows the calculated area percentages of dense Aβ plaques (upper graph) and diffuse Aβ plaques (lower graph) in the brains of WT (B6SJLF1 / J) and 5×FAD mice. The area percentages in each brain were averaged over four brain sections at bregma −0.25, −1.30, −2.35, and −3.40 mm (N = 10, 13, 8, 8, 10, 8, 9, 9). In Figure 15B, results of one-way ANOVA followed by Sidak test: ** indicates p<0.01 vs. vehicle (Veh), *** indicates p<0.001 vs. vehicle (Veh), # indicates p<0.05 for the same dose of rfmAb-1 vs. 17P04, and ## indicates p<0.01 for the same dose of rfmAb-1 vs. 17P04. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present disclosure relates to and provides novel anti-Aβ protofibril / oligomer antibodies and antibody fragments having fully human variable regions that preferentially bind soluble Aβ protofibrils / oligomers, and the anti-Aβ protofibril / oligomer antibodies and antibody fragments are capable of eliciting antibody-dependent cell-mediated phagocytosis (ADCP) in cells, including microglial cells, exposed to the anti-Aβ protofibril / oligomer antibodies and antibody fragments bound to Aβ protofibrils / oligomers. Furthermore, the anti-Aβ protofibril / oligomer antibodies and antibody fragments are capable of penetrating brain tissue after administration to a mammalian subject and reducing the levels of soluble Aβ oligomers / protofibrils and insoluble Aβ fibrils / plaques, including both dense and diffuse plaques, measured in the brain tissue after administration to the subject. Both Aβ oligomers and Aβ protofibrils are soluble and neurotoxic Aβ aggregates formed by monomers that are presumed to differ in structure and molecular weight; however, without wishing to be bound by a particular model or mechanism of action, it is noted that the present disclosure relates to and provides novel anti-Aβ protofibril / oligomer antibodies and antibody fragments that preferentially bind soluble Aβ protofibrils / oligomers and provide a therapeutic effect related to the reduction of soluble Aβ oligomer / protofibril levels in one or more tissues after administration to a subject.

[0044] Terms / Definitions

[0045] Scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art unless otherwise defined. Use of singular terms ("a" or "an" or "the" or other uses of terms in the singular) includes plural references and plural terms include the singular unless the context clearly dictates otherwise. Thus, for example, a reference to an "antibody" includes "one or more" antibodies or "a plurality" of such antibodies. All publications mentioned herein are hereby incorporated by reference in their entirety.

[0046] Generally, the terms and techniques of molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry, and recombinant DNA technology available to those skilled in the art can be adapted for use with the antibodies, antigen-binding fragments, compositions, and methods disclosed herein. The techniques and procedures described herein are generally carried out according to conventional methods known in the art, as described in various general and more specific references, particularly Sambrook et al. (1989) MOLECULAR CLONDING: A LABORATORY MANUAL (2nd ed., 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). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein, unless otherwise specified herein. Techniques and methods for preparing and formulating pharmaceuticals and treating subjects are described herein using conventional terminology.

[0047] The terms "antibody" and "antibody fragment" refer in their broadest sense to a polypeptide or combination of polypeptides that recognizes and binds to an antigen through one or more immunoglobulin variable regions, where the immunoglobulin variable regions may be natural or non-natural, e.g., as a result of genetic engineering, chimerization, humanization, optimization, CDR-grafting, or affinity maturation. The antibodies or antibody fragments disclosed herein comprise at least sufficient complementarity-determining regions (CDRs) interspersed with framework regions (FRs) for an antibody to recognize and bind to an antigen. Depending on the desired function, the antibodies or antibody fragments disclosed herein may also comprise additional peptides or other moieties, such as sufficient constant region (also called constant domain) structure / sequence to elicit a desired response, e.g., ADCP in effector cells, or regions supporting labels, tags, linkers, conjugation, etc., that allow the antibody or antibody fragment to be detected, imaged, conjugated, tagged, crosslinked, immobilized, removed, etc.

[0048] The antibody or antibody fragment may be, but is not limited to, at least one of a monoclonal antibody, a recombinant monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a Fab fragment, a single-chain variable fragment (scFv), an aptamer, a single-domain antibody (VHH or nanobody), a recombinant antibody, a modified antibody having peptides / other moieties appended to the antibody and / or additional amino acids added to the N- or C-terminus, or other antigen-binding fragment or variant. 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 particular source or species, while the remaining portions of the HC and / or LC are derived from a different source or species; for example, the target binding region (usually the variable region) will be derived from one species (e.g., human, non-human primate, or mouse) and the constant region (particularly the Fc) is derived from a different species. Chimeric antibodies can be produced by sources genetically engineered to produce antibodies with defined variable and constant regions, for example, by mice genetically engineered to express entire human variable immunoglobulin regions, producing antibodies with fully human variable regions and constant regions that may include mouse constant region sequences, e.g., mouse Fc, and / or by cells genetically engineered to produce antibodies with similar defined variable region / constant region formats. While the present disclosure seeks to provide antibodies with fully human variable regions, it is understood that in some embodiments, the antibodies may be natural chimeric antibodies produced by cells derived from mice (Mus musculus) genetically engineered to produce antibodies with fully human variable regions. In other embodiments, the antibodies may be recombinant antibodies produced by cells expressing one or more constructs encoding antibodies with fully human variable regions in a defined variable region / constant region format, such as fully human or chimeric antibodies, where the constant region components, including Fc, are derived from a class / subclass (isotype) selected for the particular use and target of the antibody.As used herein, the term "humanized antibody" refers to an antibody or antibody variant derived from a non-human antibody, typically a mouse monoclonal antibody, having a non-human variable region, in which the CDRs from the parent non-human antibody are grafted (fused) into a framework comprising a variable region derived from a human immunoglobulin framework, specifically an acceptor human framework or a human consensus framework. Techniques and principles for designing, producing, 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-Jun 4;321(6069):522-5; Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33). It will be appreciated that modifications can be made to the acceptor framework in multiple places to develop humanized antibodies with improved characteristics, e.g., higher affinity for the target, lower clearance, lower toxicity, etc., according to the desired use. The constant region (constant domain) of an antibody or antibody fragment can be derived from one of five major antibody classes: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, each of which is well characterized and known to provide functional specialization. Modified versions of each of these classes and isotypes are readily identifiable and are within the scope of this disclosure. While all immunoglobulin classes are within the scope of this disclosure, this disclosure will be largely directed to the IgG class of immunoglobulin molecules.

[0049] The anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein can be whole (intact, full-length) antibodies, single-chain antibodies, or antibody fragments having one or two chains, and can be natural or non-naturally occurring. The antibodies or antibody fragments disclosed herein comprise at least sufficient complementarity-determining regions (CDRs) interspersed with framework regions (FRs) for the antibody to recognize and bind to an antigen, preferably Aβ protofibrils / oligomers. The anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein may have part or all of a variable region portion comprising at least a sufficient amount of fully human heavy chain variable region polypeptide (VH) and a sufficient amount of fully human light chain variable region polypeptide (VL), which together form a structure having a binding domain that interacts with an antigen, preferably soluble Aβ protofibrils / oligomers. It should be understood that the variable region of the anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein can comprise two VH-VL structures. The anti-Aβ protofibril / oligomer antibodies disclosed herein may be full-length antibodies, intact antibodies, or native antibodies, or equivalent terms that are understood to refer to polypeptides comprising two full-length heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, which form constant and variable regions. It should be understood that the anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein, particularly the anti-Aβ protofibril / oligomer antibody fragments disclosed herein, may comprise an HC polypeptide and are not necessarily considered to be full-length HCs, particularly in the constant region, but have sufficient structure / sequence for a desired function, e.g., a sufficient constant region structure / sequence to elicit a desired response, specifically, a sufficient Fc structure / sequence to elicit ADCP in effector cells, or a sufficient constant region structure / sequence to support a label, tag, linker, conjugation, etc. that allows for detection, imaging, tagging, conjugation, crosslinking, immobilization, removal, etc., of the antibody or antibody fragment.

[0050] In the anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein, each of the VH and VL regions can be further divided into CDR regions characterized by hypervariability, which are typically interspersed with more highly conserved FR regions. Each of the VH and VL regions typically consists of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibody constant region may mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system and the classical complement system, and / or may elicit effector cell function. Typically, the anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein contain at least heavy chain (HC)-CDR1, HC-CDR2, and HC-CDR3 sequences, as well as light chain (LC)-CDR1, LC-CDR2, and LC-CDR3 sequences. The VH and VL amino acid sequences of non-limiting embodiments of anti-Aβ protofibril / oligomer antibodies or antibody fragments are disclosed in Table 2 (VH) and Table 3 (VL), with the CDRs identified using the IMGT numbering system (imgt.org) underlined in the VH and VL regions, respectively, and shown as separate sequences. Nucleotide sequences encoding the VH and VL of non-limiting embodiments of anti-Aβ protofibril / oligomer antibodies or antibody fragments are disclosed in the Sequence Listing, which is incorporated herein by reference. An antibody may contain fewer CDR sequences, as long as the antibody is capable of recognizing and binding to an antigen.

[0051] The anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein may be variants containing at least one altered CDR or framework sequence, and the CDR and / or framework sequence may be optimized by mutating the nucleic acid molecule encoding such framework sequence. The anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein may have HC and LC portions independently obtained from different sources. Techniques for generating variants include, but are not limited to, conservative amino acid substitution, computer modeling, screening of candidate polypeptides alone or in combination, and codon optimization. It should be understood that those skilled in the art can generate antibody variants as needed. The anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein may be fragments, provided that the fragments retain the ability to induce ADCP upon binding to Aβ protofibrils / oligomers. The antigen-binding function of an antibody can be performed by fragments such as Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region, Fd fragments consisting of the VH and CH1 domains, single-chain variable fragments (scFv) consisting of the VL and VH domains of a single arm of an antibody, single-domain antibody (dAb) fragments consisting of the VH domain, and isolated CDRs (VHH, nanobody), or aptamers. Antigen-binding moieties can be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). Antigen-binding portions of antibodies can be grafted onto polypeptide-based scaffolds to form monobodies (see, eg, US Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0052] The terms including "conditions associated with Aβ protein aggregation" or "conditions associated with Aβ protein aggregation and deposition" or "Aβ peptide-related diseases" are understood to refer to a group of conditions, diseases, and disorders associated with Aβ plaque formation, specifically extracellular Aβ plaque formation. Neurological conditions associated with Aβ protein aggregation and deposition include, but are not limited to, Alzheimer's disease (AD); mild cognitive impairment (MCI); dementia with Lewy bodies; Down's syndrome; hereditary cerebral hemorrhage with amyloidosis; and other diseases based on or related to amyloid-like proteins, such as cerebral amyloid angiopathy, primary and secondary systemic amyloidosis, familial amyloid polyneuropathy 1, progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, Parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), inclusion body myositis (IBM), type II diabetes, and senile cardiac amyloidosis; as well as various ocular diseases, including glaucoma, macular degeneration, drusen-associated optic neuropathy, and cataracts due to beta-amyloid deposition.

[0053] In the present disclosure, the terms "Aβ protofibrils / oligomers" and "Aβ protofibrils and / or oligomeric Aβ," including similar terms, are used with the understanding that Aβ oligomers and Aβ protofibrils are recognized as soluble, neurotoxic Aβ formed by monomers and are expected to exist in a sample, possibly in a variety of different structures, conformations, associations, and molecular weights (e.g., protofibrils >75 kDa, whereas oligomers <75 kDa). Therefore, "Aβ protofibrils / oligomers" can encompass Aβ protofibrils, or Aβ oligomers, or a mixture of Aβ protofibrils and Aβ oligomers, or a complex (association) of Aβ protofibrils and Aβ oligomers. Therefore, terms such as "detecting Aβ protofibrils / oligomers" are understood to encompass detecting any or all of Aβ protofibrils, or Aβ oligomers, or both Aβ protofibrils and Aβ oligomers, or complexes (associations) of Aβ protofibrils and Aβ oligomers.

[0054] In the present disclosure, "preferential binding to Aβ protofibrils / oligomers" or "preferentially binds soluble Aβ protofibrils / oligomers" or similar terms are understood to indicate selectivity or affinity for Aβ protofibrils / oligomers, as measured by common methods known in the art, including those described herein. Approximately 50% of maximal binding (calculated EC 50 The calculated concentration at which the Aβ binding occurs can often be used as an estimate of affinity. Because Aβ protein in a sample can exist in multiple conformations of Aβ secondary structure (monomer, oligomer / protofibril, fibril, etc.), it is understood that "preferential binding" or "selectivity" or similar terms refer to significantly higher binding of an antibody or antibody fragment to Aβ oligomers / protofibrils compared to binding of the same antibody or antibody fragment to Aβ monomers in a sample. A non-limiting approach to determining selectivity for Aβ oligomers / protofibrils over Aβ monomers is to use IC20 measured in a competitive ELISA assay.50 EC measured by capture ELISA assay against oligomers 50 The ratio can be calculated as the ratio of monomer to soluble Aβ oligomers / protofibrils. This ratio is used to identify and rank anti-Aβ protofibril / oligomer antibodies and antibody fragments according to their level of preferential binding to soluble Aβ oligomers / protofibrils.

[0055] A "subject" is a mammal, including, but not limited to, primates (e.g., humans and non-human primates such as monkeys), mammals commonly used in research, such as rabbits and rodents (e.g., mice and rats), and livestock animals (e.g., cows, sheep, cats, dogs, pigs, llamas, and horses). In certain embodiments, the subject is a human. The phrases "to a subject in need thereof" or "to a patient in need thereof" or "to a patient in need of treatment" or "to a subject in need of treatment" may include subjects who would benefit from administration of the anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein for the treatment, prevention, diagnosis, screening, or monitoring of a condition associated with Aβ protein aggregation, specifically AD. It should be understood that administration of an anti-Aβ protofibril / oligomer antibody or antibody fragment encompasses administration to a "subject in need thereof," which may be interpreted as referring to a subject known or suspected to have a condition associated with Aβ protein aggregation, specifically AD. The anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein can be administered to subjects known or suspected of having a condition associated with Aβ protein aggregation, specifically AD, for therapeutic, preventative, or prophylactic purposes, including, but not limited to, for treatment, screening, diagnostic, monitoring, research purposes, or to achieve a result other than treating a disorder. Furthermore, it should be understood that the anti-Aβ protofibril / oligomer antibodies or antibody fragments can be administered to subjects not known or suspected of having a condition associated with Aβ protein aggregation, including, but not limited to, for prevention or prophylactic purposes, screening, diagnostic, monitoring, research purposes, or to achieve a result other than treating a disorder.

[0056] composition

[0057] Anti-Aβ protofibril / oligomer antibodies and antibody fragments that selectively bind to soluble Aβ protofibrils / oligomers induce ACDP in effector cells, penetrate into brain tissue, reduce the levels of soluble Aβ protofibrils / oligomers in brain tissue, reduce insoluble Aβ plaques, including dense plaques and diffuse plaques, and reduce plaque burden in brain tissue.

[0058] Antibodies and antigen-binding fragments are provided that exhibit selectivity and preferential binding for soluble Aβ protofibrils / oligomers. Specifically, anti-Aβ antibodies and antigen-binding fragments are provided that have sufficient constant region sequence, including sufficient Fc structure / sequence, such that complexes of the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to Aβ protofibrils / oligomers can elicit ADCP in phagocytic effector cells, thereby phagocytosing and therefore removing the complexes of the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to Aβ protofibrils / oligomers from the solution surrounding the effector cells. Without wishing to be bound by a particular mechanism of action or hypothesis, ADCP-mediated removal of the complexes of the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to Aβ protofibrils / oligomers from the solution surrounding the effector cells can be achieved, resulting in removal of soluble Aβ protofibrils / oligomers from the solution. It should be understood that ADCP-mediated removal of anti-Aβ protofibril / oligomer antibody or antibody fragment complexes bound to Aβ protofibrils / oligomers from the solution surrounding effector cells can occur in vivo, ex vivo, or in vitro. In vivo ADCP-mediated removal of anti-Aβ protofibril / oligomer antibody or antibody fragment complexes bound to Aβ protofibrils / oligomers can occur in, among other places, blood, lymph, cerebrospinal fluid (CSF), neural tissue, or the brain. Thus, in vivo ADCP-mediated removal of anti-Aβ protofibril / oligomer antibody or antibody fragment complexes bound to Aβ protofibrils / oligomers can reduce the level of soluble Aβ protofibrils / oligomers in, among other places, blood, lymph, cerebrospinal fluid (CSF), neural tissue, or the brain. ADCP-mediated removal of anti-Aβ protofibril / oligomer antibody or antibody fragment complexes bound to Aβ protofibrils / oligomers ex vivo or in vitro can occur in samples such as blood, lymph, cerebrospinal fluid (CSF), or other samples collected from a subject, and can reduce the levels of soluble Aβ protofibrils / oligomers in such samples.

[0059] The anti-Aβ antibodies and antigen-binding fragments provided herein having fully human variable regions are expected to have a lower risk of inducing an immune response and result in a reduced risk of adverse reactions or undesirable side effects when administered to a human subject. In contrast, humanized antibodies derived from non-human antibodies, such as lecanemab, a humanized IgG1 monoclonal antibody that preferentially binds Aβ protofibrils / oligomers, may be expected to have a relatively higher risk of adverse reactions or undesirable side effects after administration.

[0060] Exemplary embodiments of antibodies and antigen-binding fragments that preferentially bind to soluble Aβ protofibrils / oligomers and elicit ADCP include, but are not limited to, the antibodies shown in the Examples and Tables 1, 2, 3, 4, and 5. In certain embodiments, Aβ 1-42Anti-Aβ protofibril / oligomer antibodies with fully human variable regions produced by transgenic mice immunized with protofibrils were recovered from hybridoma supernatants and screened as described in Example 1 to generate chimeric monoclonal antibodies with fully human F(ab')2 and mouse CH2 and CH3 domains. In a specific embodiment, 33 chimeric monoclonal antibodies with fully human F(ab')2 and mouse IgG1 CH2 and CH3 domains listed in Table 1 were evaluated for their ability to preferentially bind to soluble Aβ protofibrils / oligomers and induce ADCP; the VH and VL sequences of 15 antibodies of interest are shown in Tables 2 and 3. In certain embodiments, anti-Aβ protofibril / oligomer antibodies were prepared in different formats for different practical and potential uses and evaluated for their ability to preferentially bind soluble Aβ protofibrils / oligomers and elicit ADCP, including a chimeric monoclonal antibody having fully human F(ab')2 and mouse IgG1 CH2 and CH3 domains (i.e., chimeric human F(ab')2 / mouse IgG1 antibody) with the variable region sequences shown in Tables 2 and 3 and tested as shown in Table 1; an antibody reformatted as a fully human IgG1 lambda antibody and tested as described in Example 5 and shown in Table 4; and an antibody reformatted as mouse IgG2a CH2-CH3 / (fully human F(ab')2 and mouse IgG2a CH2 and CH3 domains), tested as described in Example 5 and shown in Table 5, and used in Examples 6 and 7.

[0061] The sequence and format of anti-Aβ protofibril / oligomer antibodies and antibody fragments can be modified to develop antibodies or fragments with desired properties. In one non-limiting embodiment, anti-Aβ protofibril / oligomer antibodies or antibody fragments with desired properties for a specific use can be developed using an appropriate variable region framework including a VH comprising an HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO: 42), an HC CDR2 having the amino acid sequence ISSRRTYI (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) comprising 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), in combination with a constant region sequence comprising an Fc sequence selected as appropriate for the specific use. In another non-limiting embodiment, an anti-Aβ protofibril / oligomer antibody or antibody fragment with desired properties for a specific use can be developed using an appropriate variable region framework comprising a VH comprising an H CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO: 62), an H CDR2 having the amino acid sequence IRSKANSYAT (SEQ ID NO: 63), and an H CDR3 having the amino acid sequence TSHAPNFDAFDI (SEQ ID NO: 64), and a VL comprising an LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 67), an LC CDR2 having the amino acid sequence DNS, and an LC CDR3 having the amino acid sequence GTWDSSLSTYV (SEQ ID NO: 69), in combination with a constant region sequence comprising an Fc sequence selected as appropriate for the specific use.In another non-limiting embodiment, an anti-Aβ protofibril / oligomer antibody or antibody fragment with desired properties for a specific use can be developed using an appropriate variable region framework comprising a VH comprising an HC CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO: 72), an HC CDR2 having the amino acid sequence IRSKVNSYAT (SEQ ID NO: 73), and an HC CDR3 having the amino acid sequence TSHAAPIFDAFDI (SEQ ID NO: 74), and a VL comprising an LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 77), an LC CDR2 having the amino acid sequence DNS, and an LC CDR3 having the amino acid sequence GTWDSSLSTYF (SEQ ID NO: 79), in combination with a constant region sequence comprising an Fc sequence selected as appropriate for the specific use described above. In another non-limiting embodiment, an anti-Aβ protofibril / oligomer antibody or antibody fragment with desired properties for a specific use can be developed using an appropriate variable region framework comprising a VH comprising an HC CDR1 having the amino acid sequence GFTFSNAW (SEQ ID NO: 132), an HC CDR2 having the amino acid sequence IKSKTDGGTR (SEQ ID NO: 133), and an HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 134), and a VL comprising an LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO: 137), an LC CDR2 having the amino acid sequence RNN, and an LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 139), in combination with a constant region sequence comprising an Fc sequence selected as appropriate for the specific use, as described above.In another non-limiting embodiment, an anti-Aβ protofibril / oligomer antibody or antibody fragment with desired properties for a specific use can be developed using an appropriate variable region framework comprising a VH comprising an HC CDR1 having the amino acid sequence GFSFSNAW (SEQ ID NO: 2), an HC CDR2 having the amino acid sequence IKSKTDGGTI (SEQ ID NO: 3), and an HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 4), and a VL comprising an LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO: 7), an LC CDR2 having the amino acid sequence RNN, and an LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 9), in combination with a constant region sequence comprising an Fc sequence selected as appropriate for the specific use. It should be understood that a "suitable variable region framework" is an appropriate human variable region framework that can be selected or genetically modified for a specific property or use.

[0062] Anti-Aβ protofibril / oligomer antibodies and antibody fragments capable of penetrating brain tissue are provided. Specifically, anti-Aβ protofibril / oligomer antibodies and antibody fragments having fully human variable regions capable of penetrating brain tissue are provided. It should be understood that brain penetrance, i.e., the ability to cross the blood-brain barrier, enables an antibody or antibody fragment to act on a target within brain tissue. Without wishing to be bound by a particular mechanism of action or hypothesis, ADCP-mediated removal of complexes of anti-Aβ protofibril / oligomer antibodies or antibody fragments bound to Aβ protofibrils / oligomers in brain tissue can have effects that may include reducing the level of soluble Aβ protofibrils / oligomers in brain tissue. Without wishing to be bound by a particular mechanism of action or hypothesis, ADCP in brain tissue elicited by target-bound anti-Aβ protofibril / oligomer antibodies or antibody fragments can have effects that may include reducing the amount of Aβ plaques in brain tissue. It is understood that reducing the amount of Aβ plaques can include removing Aβ plaques.

[0063] Exemplary embodiments of anti-Aβ protofibril / oligomer antibodies and antibody fragments capable of penetrating brain tissue, tested as described in Example 6, include, but are not limited to, 18P01A(mIgG2a) (HC amino acid sequence of SEQ ID NO: 155; LC amino acid sequence of SEQ ID NO: 156) comprising an 18P01A fully human VH having the amino acid sequence of SEQ ID NO: 61 and an 18P01A fully human VL having the amino acid sequence of SEQ ID NO: 66; and 17P04A(mIgG2a) (HC amino acid sequence of SEQ ID NO: 151; LC amino acid sequence of SEQ ID NO: 152) comprising a 17P04A fully human VH having the amino acid sequence of SEQ ID NO: 41 and a 17P04A fully human VL having the amino acid sequence of SEQ ID NO: 46. Both embodiments demonstrated acceptable half-lives in serum after dose administration (FIG. 9) and were detectable in brain tissue after dose administration (FIG. 10), with 18P01A (mIgG2a) having a brain penetrance of 0.3% and a brain tissue half-life of 77.2 hours, and 17P04A (mIgG2a) having a brain penetrance of 5.5% and a brain tissue half-life of 92.4 hours. Additional anti-Aβ protofibril / oligomer antibodies and antibody fragments presented herein can be evaluated for serum half-life, brain penetrance, and brain half-life.

[0064] Anti-Aβ protofibril / oligomer antibodies and antibody fragments are provided that can reduce the level of soluble Aβ oligomers / protofibrils and the level of insoluble Aβ plaques, including dense plaques and diffuse plaques, in the brain of a mammalian subject after administration to the subject. Specifically, anti-Aβ protofibril / oligomer antibodies and antibody fragments having fully human variable regions are provided that can reduce the level of soluble Aβ oligomers / protofibrils and the level of insoluble Aβ plaques, including dense plaques and diffuse plaques, in the brain of a mammalian subject after administration to the subject. Without wishing to be bound by a particular mechanism of action or hypothesis, reducing the level of insoluble Aβ plaques in brain tissue can include reducing, preventing, or delaying the aggregation and deposition of Aβ protein as insoluble Aβ plaques. Using animal models of conditions associated with Aβ protein aggregation, we measured the effects of anti-Aβ protofibril / oligomer antibodies and antibody fragments on soluble Aβ oligomers / protofibrils and reduced the level of insoluble Aβ plaques in brain tissue after administration. In one non-limiting, exemplary embodiment (Example 7) using 5×FAD mice as an AD mouse model to evaluate the long-term efficacy of anti-Aβ protofibril / oligomer antibodies, the results showed that administration of 17P04A (mIgG2a) reduced the level of soluble Aβ oligomers / protofibrils and insoluble Aβ plaques, including dense and diffuse plaques, in the brains of 5×FAD mice compared to the levels of soluble Aβ oligomers / protofibrils and insoluble Aβ plaques in control 5×FAD mice injected with vehicle (PBS) over the same time period (Figure 13). 17P04A (mIgG2a) (HC SEQ ID NO: 151, LC SEQ ID NO: 152) exerted a dose-dependent effect on soluble Aβ oligomers / protofibrils ( FIG. 13 ) and insoluble Aβ plaques ( FIG. 15B ), providing an example of how parameters such as antibody concentration, administration frequency, and duration of antibody treatment can be evaluated to find optimal conditions to maximize therapeutic efficacy and avoid undesirable side effects.Additional anti-Aβ protofibril / oligomer antibodies and antibody fragments provided herein can be evaluated for their effect on soluble Aβ oligomers / protofibrils and insoluble Aβ plaques in brain tissue after administration to a subject.

[0065] Compositions are provided that include the anti-Aβ protofibril / oligomer antibody or antibody fragment of the present invention and a pharmaceutically acceptable carrier or excipient suitable for the intended use of each composition. 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 compatible with the method of administration.

[0066] The anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered to a subject by any suitable means, including, but not limited to, injection or parenteral administration. Parenteral administration can include intramuscular, intravenous, intraarterial, intraperitoneal ("ip"), subcutaneous, intraspinal (including epidural or intrathecal), intraocular, intracerebral, intraventricular, intracardiac, intradermal, intraarticular, intralymphatic, or intraosseous administration. The anti-Aβ oligomer antibodies and antibody fragments disclosed herein are intended for parenteral administration, specifically intraperitoneal administration, into the circulatory system. The anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered using a device, as a depot, or in the form of a sustained-release formulation (e.g., semipermeable matrices of solid hydrophobic polymers containing the antibody, or microcapsules) that allow for slow, measured, and / or localized delivery. The anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be formulated and administered using colloid drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in the form of macroemulsions.

[0067] method

[0068] Methods are provided for treating, preventing, diagnosing, screening, or monitoring conditions associated with Aβ protein aggregation, specifically AD, using an effective amount of the anti-Aβ protofibril / oligomer antibodies or antibody fragments disclosed herein. While not wishing to be bound by a particular mechanism of action, methods are provided for precisely targeting and inhibiting the amount and effects of Aβ protofibrils / oligomers, specifically soluble Aβ protofibrils / oligomers, using the anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein. Methods for precisely targeting and inhibiting soluble Aβ protofibrils / oligomers can include methods using a route of administration to a targeted location or system involved in the disorder, methods using administration timed to a targeted critical period, and combinations of these methods.

[0069] Methods are provided for treating conditions associated with Aβ protein aggregation, specifically AD, by removing soluble Aβ protofibrils / oligomers from tissues, body fluids, or samples. Without wishing to be bound by a particular mechanism of action, methods are provided for treating conditions associated with Aβ protein aggregation by removing or reducing the levels of precursors of Aβ protein aggregation, specifically by ADCP-mediated removal of anti-Aβ protofibril / oligomer antibody or antibody fragment complexes bound to Aβ protofibrils / oligomers. Methods and compositions are provided for treating disorders associated with Aβ protein aggregation, specifically AD, that can be used to remove soluble Aβ protofibrils / oligomers from blood, lymph, cerebrospinal fluid (CSF) collected from a subject or in a sample of interest. It should be understood that in vivo methods for treating conditions associated with Aβ protein aggregation include administering a therapeutic amount of an anti-Aβ protofibril / oligomer antibody or antibody fragment to a subject.

[0070] The methods and compositions provided herein enable strategic timing of treatment. While not wishing to be bound by a particular mechanism of action, the anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered after a screening or diagnosis indicating that a subject has or is suspected of having a condition associated with Aβ protein aggregation, specifically AD. Thus, the anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered at a time when the prevention or further development of a condition associated with Aβ protein aggregation, specifically AD, may be halted or possibly reversed. While not wishing to be bound by a particular mechanism of action, the anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered to subjects identified as at risk of having or developing a condition associated with Aβ protein aggregation, specifically AD. Thus, the anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered at a time when the onset or further development of a condition associated with Aβ protein aggregation, specifically AD, may be prevented or halted. Additionally, the anti-Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered prophylactically.

[0071] The methods and compositions provided herein allow for control of the amount of therapeutic or prophylactic agent present at one or more points during treatment. Without wishing to be bound by a particular mechanism of action, the Aβ protofibril / oligomer antibodies and antibody fragments disclosed herein can be administered and effects monitored, for example, by measures of response such as edema or inflammation, levels of soluble Aβ protofibrils / oligomers or other Aβ conformations (monomers, fibrils, plaques), or clinical measures of cognitive function. While not wishing to be bound by a particular mechanism of action, a dose or "effective amount" sufficient to produce at least one desired effect (e.g., reduction in soluble Aβ protofibril / oligomer levels, reduction in Aβ plaque levels, or improvement in cognitive function) with little or tolerable undesirable effects (e.g., edema or inflammation) may depend on factors specific to the subject in need thereof; therefore, treatment may include determining the dose of anti-Aβ protofibril / oligomer antibody or antibody fragment necessary to constitute an effective amount for use in a subject, and administering said effective amount of the anti-Aβ protofibril / oligomer antibody or antibody fragment to the subject.

[0072] Methods are provided for diagnosing and screening for at least one condition associated with Aβ protein aggregation in a subject, and for detecting the presence and, optionally, the amount of soluble Aβ protofibrils / oligomers in a sample from the subject, by using at least one anti-Aβ protofibril / oligomer antibody or antibody fragment disclosed herein. The sample can be a soluble fraction or a fixed tissue sample. It should be understood that the diagnostic or screening method includes comparing a result, such as the amount of soluble Aβ protofibrils / oligomers in the sample, with at least one reference value related to the presence and / or amount of soluble Aβ protofibrils / oligomers in another sample. The reference value can be based on a sample from another subject who may or may not be considered to have a condition associated with Aβ protein aggregation, specifically AD, or can be a value in a sample from the same subject taken at a different time point.

[0073] A method for producing anti-Aβ protofibril / oligomer antibodies or antibody fragments with fully human variable regions that preferentially bind soluble Aβ protofibrils / oligomers using a functional screening approach. The method includes immunizing a mammal that produces antibodies having human variable regions by introducing a preparation of purified Aβ protofibrils (which may contain oligomers) into the mammal; harvesting enriched B cells and fusing the B cells with a myeloma fusion partner to generate hybridomas; recovering antibodies from each hybridoma; performing a primary screen of the recovered antibodies from each hybridoma, which screens the recovered antibodies by measuring binding to Aβ peptide in an ELISA-based assay; identifying and recovering any antibodies that have a sufficient level of detectable binding to Aβ peptide; and performing a conformational screen of each recovered antibody after the primary screen, which screens the recovered antibodies by measuring competition of Aβ monomers and Aβ protofibrils for binding to the recovered antibodies using a competitive ELISA, measuring the direct binding of each recovered antibody to Aβ monomers, Aβ protofibrils, and Aβ fibrils by ELISA, and selecting each antibody that exhibits higher affinity for protofibrils over monomers in the competitive ELISA. It should be understood that the mammals described above are genetically modified to produce antibodies with human regions, with mice typically being genetically modified for such functions. Each anti-Aβ protofibril / oligomer antibody or antibody fragment can be formatted for a particular test or function, for example, by combining a constant region, including an Fc region, with a desired function and / or species compatibility, with a variable region (VH / VL) characterized by a desired level of selectivity and preferential binding to soluble Aβ protofibrils / oligomers.Further screening and characterization steps are provided, including measuring the ability of the anti-Aβ protofibril / oligomer antibody or antibody fragment to trigger ADCP-mediated removal of anti-Aβ protofibril / oligomer antibody or antibody fragment complexes bound to Aβ protofibrils / oligomers, and assessing brain penetrance of the anti-Aβ protofibril / oligomer antibody or antibody fragment and the subsequent reduction in plaque levels in brain tissue, which indicates its ability to clear plaques. Suitable Fc regions for use with the anti-Aβ protofibril / oligomer antibodies or antibody fragments provided herein can be all or part of the Fc regions of, but are not limited to, mouse IgG1, human IgG1, mouse IgG2a, human IgG3, and mouse IgG2b.

[0074] In one non-limiting exemplary embodiment, results of a long-term efficacy study show that treatment with an antibody comprising a fully human VH comprising a 17P04A HC CDR1 having the sequence GFTLSSFS (SEQ ID NO: 42), a 17P04A HC CDR2 having the sequence ISSRRTYI (SEQ ID NO: 43), and a 17P04A HC CDR3 having the sequence ARGGYIGSPNAYDI (SEQ ID NO: 44), and a fully human VL comprising a 17P04A LC CDR1 having the sequence TGAVTSDYY (SEQ ID NO: 47), a 17P04A LC CDR2 having the sequence SAS, and a 17P04A LC CDR3 having the sequence LLYYGGAWV (SEQ ID NO: 49) efficiently and effectively clears soluble Aβ oligomers / protofibrils and insoluble Aβ fibrils / plaques (including both dense and diffuse plaques) in the brain, thereby reducing plaque burden in vivo in an AD disease model.

[0075] The following examples are offered to illustrate, but not to limit, the claimed invention. Example

[0076] Example 1: Generation and identification of antibodies that bind amyloid beta (Aβ) oligomers / protofibrils

[0077] The generation of novel monoclonal antibodies against amyloid beta (Aβ) oligomers was performed under contract with AlivaMab Discovery Services, LLC (San Diego, CA). AMX-KL mice, a transgenic mouse strain that produces antibodies with fully human F(ab')2 domains containing human VH and VL, human CL and CH1, and mouse CH2 and CH3 domains, were licensed from Ablexis, LLC (ablexis.com, San Diego, CA) and were used for therapeutic antibody discovery. AMX-KL mice were immunized with freshly prepared Aβ protofibrils. Aβ 1-42Protofibrils were prepared according to the protocol detailed below. Chromatographic profiles are shown in Figure 1. Enriched B cells obtained from the spleens and lymph nodes of mice with sufficient plasma titers were fused with myeloma fusion partners to elicit downstream antibody recovery and screen for activity. A series of screening and characterization steps employed enzyme-linked immunosorbent assay (ELISA) techniques. First, 15,662 hybridoma supernatants were subjected to primary screening for binding to Aβ peptide using an ELISA-based assay. The top 186 hits with binding affinity to Aβ peptide of >6,900,000 RLU were selected and advanced to conformational screening, which involved measuring competition between Aβ monomers and protofibrils and direct binding to monomers, protofibrils, and fibrils using ELISA. Thirty-three hits that showed higher affinity for protofibrils than monomers in competitive ELISA were selected and purified from the conformational screening. After purification, each of the 33 monoclonal antibodies (mAbs) was characterized and ranked in a panel of in vitro assays (results are shown in Table 1), and the five best mAbs were identified as lead candidates. Fifteen antibodies were subcloned to obtain variable region sequences (see Tables 2 and 3). The five lead candidates were purified and characterized. The five lead candidates were then reformatted for further characterization and development as (a) fully human antibodies and (b) chimeric antibodies with different mouse isotypes.

[0078] Preparation of different Aβ conformations

[0079] 1 mg of 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 10× PBS (final pH approximately 8.8) and incubated at 37°C for 2 hours to allow protofibril formation. 1-42The aggregation state of Aβ was monitored by a thioflavin T assay (Sigma, T3516, sigmaaldrich.com, 10 μM thioflavin T in the sample solution). This method produced a protofibril / monomer mixture containing approximately 70% protofibrils and approximately 30% monomers, as shown in Figure 1, which presents the results of size exclusion chromatography (Superdex 200 Increase 10 / 300 GL, Cytiva 28-9909-44) of the mixture. This protofibril / monomer mixture was freshly prepared on each day of immunization. The amount of Aβ in the mixture was significantly reduced. 1-42 The experiment was carried out under the assumption that the monomer may not elicit an immune response due to its small size (molecular weight 4514 Da).

[0080] Purified Aβ for antibody screening 1-42 To prepare protofibrils, fractions of the protofibril peak during size exclusion chromatography were collected and stored at −80°C. 1-42 Protofibril stocks are stable for at least 2 months at -80°C and can withstand multiple freeze-thaw cycles. 1-42 Although the monomer is unstable, it is suitable for antibody screening. Therefore, Aβ lacking the two C-terminal amino acid residues was used. 1-40 Monomer (rPeptide, A-1157-2) was used as the monomer conformation for antibody screening. 1-40 The Aβ monomer is stable for at least 24 hours at 4°C and can be stored at -80°C for at least 2 months. 1-42 Fibrils were purchased as preformed fibrils from rPeptide (rPeptide, catalog number AF-1002).

[0081] Example 2: Characterization and ranking of anti-Aβ protofibrillar / oligomer monoclonal antibodies

[0082] We developed a series of ELISA-based binding assays, including direct ELISA, competitive ELISA, and capture ELISA, for hybridoma screening and mAb characterization. Furthermore, we developed an antibody-dependent cell-mediated phagocytosis (ADCP) assay to evaluate the potency of anti-Aβ antibodies to mediate Aβ protofibril uptake by microglia. Two control antibodies, the anti-Aβ protofibril / oligomer antibody Reference Antibody 1 (rfmAb-1) and the nonconformation-selective anti-Aβ antibody 6E10 (BioLegend, 803001), designated rfmAb-2, were used as references in the above assays.

[0083] direct ELISA

[0084] High-binding 96-well plates (Corning, 9018) were coated with 0.5 μM Aβ monomer, Aβ protofibril, or Aβ fibril for 2 h at 4 °C. After washing three times with 1x phosphate-buffered saline, 0.1% Tween 20 (PBST), each plate was blocked with 1% BSA in PBS for 1 h at room temperature (RT). Dilutions of Aβ antibody (3-fold dilutions ranging from 0 to 100 nM) were added to the plate. The plate was incubated for 1 h at RT and washed three times. The plate was then incubated for 1 h at RT with HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, G-21040; thermofisher.com) diluted 1:5000 in 1% BSA and washed three times. 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 stopped by adding 1 M sulfuric acid. The absorbance at 450 nm was measured in a plate reader. The results of rfmAb-1 (an oligomer / protofibril-selective anti-Aβ antibody) and rfmAb-2 (a non-conformation-selective anti-Aβ antibody) showed similar affinities for Aβ monomers and protofibrils in direct ELISA (Figure 2). This suggests that direct ELISA can measure the general affinity of antibodies for Aβ peptides, but cannot determine selectivity for different Aβ conformations.

[0085] Competitive ELISA

[0086] Aβ antibodies were preincubated with dilutions of Aβ monomer, Aβ protofibrils, or Aβ fibrils (3-fold dilution series with concentrations ranging from 0 to 3000 nM) at 4°C for 1 hour. The concentration of Aβ antibody used in the antibody-Aβ mixture was determined as the EC90 of the antibody with Aβ monomer as measured in a direct ELISA experiment. The mixture was then applied to an ELISA plate precoated with 0.5 μM Aβ monomer. The plate was incubated for 10 minutes at room temperature and washed three times with TBST. The plate was then incubated for 1 hour at room temperature 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 (Life Technologies, SB02) was added to the plate, and the reaction was stopped by adding 1 M sulfuric acid. The absorbance at 450 nm was measured in a plate reader. As shown in Figure 3, rfmAb-1 mixed with Aβ protofibrils showed low binding to Aβ monomers, whereas rfmAb-1 mixed with Aβ monomers or fibrils showed high binding to Aβ monomers (Figure 3A), indicating that rfmAb-1 has a higher affinity for Aβ oligomers than for Aβ monomers or fibrils, which is consistent with its known binding profile. As shown in Figure 3, rfmAb-2 showed similar affinity for Aβ monomers and Aβ protofibrils in competitive ELISA (Figure 3B), which is consistent with its known binding profile. These results suggest that competitive ELISA can distinguish the selectivity of anti-Aβ antibodies for different Aβ conformations.

[0087] Capture ELISA

[0088] A high-binding 96-well plate (Corning, 9018) was coated with 5 μg / mL of Aβ antibody overnight at room temperature and washed three times with PBST. Biotin-labeled Aβ was then added. 1-40Dilutions of the monomer (AnaSpec, AS-61483-01, 4-fold dilutions with concentrations ranging from 0 to 1000 nM) were applied to the plate and captured by the immobilized antibody. The plate was incubated at RT for 1 h and washed three times with PBST. The plate was then incubated with HRP-conjugated streptavidin (Thermo Fisher Scientific, SNN2004) diluted 1:5000 in 1% BSA for 20 min at RT and washed three times. TMB Stabilized Chromogen substrate solution (Life Technologies, SB02) was added to the plate, and the reaction was stopped by adding 1 M sulfuric acid. The absorbance at 450 nm was measured in a plate reader. Figure 4 shows the effect of rfmAb-1 on Aβ across the entire range of concentrations tested. 1-40 While rfmAb-2 weakly bound to Aβ monomers, it bound to Aβ in a concentration-dependent manner. 1-40 The results show strong binding to the Aβ monomer, consistent with the known affinity of each reference antibody for Aβ monomer. These results suggest that capture ELISA can determine the affinity of anti-Aβ antibodies for Aβ monomer.

[0089] Antibody-dependent cell-mediated phagocytosis (ADCP) assay

[0090] Aβ protofibrils labeled with HiLyte488 Fluorophore (AnaSpec, AS-60479-01, anaspec.com) were prepared as follows: 0.1 mg of HiLyte488-labeled Aβ was resuspended in 18 μL of 12 mM NaOH. 1-42 Monomers were prepared by resuspending 0.5 mg of unlabeled Aβ in 90 μL of 12 mM NaOH. 1-42 The labeled and unlabeled Aβ were mixed with the monomer (rPeptide, A-1167-1). Next, 680 μL of 10×PBS, pH 7.4 (Gibco, 70011-044) was added. 1-42 The mixture was incubated at 37°C for 2 hours to obtain HiLyte488-labeled Aβ. 1-42Protofibrils were formed. The protofibrils were then centrifuged at 16,000 g for 5 minutes at 4°C to remove insoluble fibrils. The supernatant was subjected to size exclusion chromatography (Superdex® 200 Increase 10 / 300 GL, Cytiva 28-9909-44), and a protofibril peak similar to that in Figure 1 was isolated.

[0091] Second, mouse microglial cell line BV-2 cells (AcceGen, ABC-TC212S, accegen.com) were seeded at a density of 60,000 cells per well in a 96-well cell culture plate and incubated overnight. Cells were pretreated with ADCP assay diluent containing HEPES-containing DMEM / F12 medium (Gibco, 11039021), 1% BSA, and 100 μg / mL fucoidan (Sigma-Aldrich, F8190) for 1 hour at 37°C before the ADCP assay. HiLyte488 oligomer (2.25 μg / mL) was mixed with a dilution series of Aβ antibody or mouse IgG2b (negative control) in ADCP assay diluent (800, 160, 32, and 6.4 ng / mL) for 30 minutes. The mixture was added to the BV-2 cells and incubated at 37°C for 1.5 hours to induce ADCP. The oligomer-antibody complexes bound to the cell surface were removed by treatment with 0.25% trypsin for 20 minutes at 4°C. Cells were gently scraped from the culture plate and transferred to a conical-bottom 96-well plate (Thermo Scientific, 249935). Cells were then rinsed twice with ice-cold cell staining buffer (BioLegend, 420201), fixed for 20 minutes in ice-cold fixation buffer (BioLegend, 420801), and rinsed again. Finally, cells were analyzed by flow cytometry for fluorescence, which reflects the amount of HiLyte488-labeled oligomer taken up by the cells.

[0092] Ranking of Aβ protofibril-binding monoclonal antibodies

[0093] The 33 Aβ protofibril-binding mAbs described above (Example 1) were tested using direct, competitive, and capture ELISAs to assess affinity for different Aβ conformations, as well as in ADCP assays for their ability to mediate Aβ protofibril uptake by microglia. The results are summarized in Table 1. In Table 1, the mAbs were compared by IC 50 EC in a capture ELISA for protofibrils 50 The antibodies were ranked based on the ratio of protofibrils to monomers, which reflects their selectivity for protofibrils over monomers. Based on the overall data, mAbs 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A were selected as lead candidates. Of these lead candidates, mAbs 18P01A, 17P04A, and 20O07A were selected as the top three antibodies based on their high Aβ protofibril selectivity, mAb 22D04A was selected for its highest ADCP activity among the candidates, and 06E17A was selected for its highest affinity for fibrils.

[0094] Table 1. Characterization and ranking of 33 anti-amyloid beta monoclonal antibodies. This list is based on IC 50 EC against protofibrils (competitive ELISA) 50 The antibodies are sorted by their selectivity for protofibrils over monomers, calculated as the ratio of monomers (capture ELISA). rfmAb-1 is the reference antibody for this study. Five antibodies in bold (18P01A, 17P04A, 20O07A, 22D04A, and 06E17A) are identified as lead candidates for further testing and development. UND indicates an undetermined value. [Table 1]

[0095] Example 3: Heavy and light chain variable region sequences of 15 anti-amyloid beta monoclonal antibodies

[0096] Sequences were obtained for the variable regions of 15 anti-Aβ mAbs of interest from Table 1, and CDRs were identified using the IMGT numbering system (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 the 15 anti-Aβ mAbs of interest from Table 1.

[0097] Table 2. HC variable region (VH) sequences of 15 mAbs of interest. CDRs 1-3 of each HC variable region are underlined in the full-length VH amino acid sequence. CDRs were identified using the IMGT numbering system. Each CDR is also listed separately and distinguished by the SEQ ID NO: assigned to the CDR. [Table 2] JPEG2026504818000003.jpg188159JPEG2026504818000004.jpg189159

[0098] Table 3. LC variable region (VL) sequences of 15 mAbs of interest. CDRs 1-3 of each LC variable region are underlined in the full-length VL amino acid sequence. CDRs were identified using the IMGT numbering system. Each CDR is also listed separately and distinguished by the SEQ ID NO: assigned to the CDR. [Table 3] JPEG2026504818000006.jpg215159JPEG2026504818000007.jpg75159

[0099] Example 4: Characterization of lead antibodies

[0100] Detailed profiles of the five lead mAbs 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A were developed and are described below.

[0101] Affinity of Aβ peptides in direct ELISA

[0102] Aβ using direct ELISA 1-42 and pyroglutamic acid-modified Aβ (Aβ p3-42 The affinity of each antibody to 0.5 μM Aβ 1-42 (rPeptide, A-1167-2) or Aβ p3-42 Plates were coated with IgG (AnaSpec, AS-29907-01) for 2 hours at 4°C. Dilutions of the antibodies (3-fold dilution series with concentrations ranging from 0 to 100 nM) were added to the plates for 1 hour at RT. Binding of the antibodies to the immobilized Aβ on each plate was detected with an HRP-labeled secondary antibody and TMB substrate, as described in Example 2. All five lead antibodies were able to bind to Aβ. 1-42 To calculate the EC 50 showed strong binding of <0.1 nM (Fig. 5A and Table 1), whereas Aβ p3-42 To calculate the EC 50 It showed weak binding of >50 nM (Fig. 5B and Table 1).

[0103] Selectivity for different Aβ conformations in competitive ELISA

[0104] The relative affinity of antibodies for Aβ monomer, Aβ protofibril, and Aβ fibril was assessed using competitive ELISA. Antibodies were subjected to competition with monomer, protofibril, or fibril on monomer-coated plates. Antibodies were preincubated with dilutions of Aβ monomer, Aβ protofibril, or Aβ fibril (3-fold dilution series starting at 3000 nM) for 1 hour at 4°C. The concentration of Aβ antibody used in the antibody-Aβ mixture was determined based on the EC of that antibody with Aβ monomer in direct ELISA. 90 The antibody-Aβ mixture was then applied to an ELISA plate coated with 0.5 μM Aβ monomer for 10 minutes at room temperature. Binding was detected with an HRP-labeled secondary antibody and TMB substrate as described in Example 2. All five lead antibodies had high affinity for protofibrils and low affinity for monomers (FIG. 6 and Table 1). The IC of the monomer 50The ratio of IC50 of the antibody to IC50 of the protofibrils was >300-fold for all five antibodies and >1000-fold for 18P01 and 17P04. The affinity of the five antibodies for fibrils was higher than for the monomers but lower than for the protofibrils. Because of the low affinity of the antibodies for the monomers, the monomer IC50 in the competitive ELISA was 50 The inventors further subjected the five antibodies to capture ELISA to obtain accurate affinities for the monomer.

[0105] Affinity of Aβ monomers in capture ELISA

[0106] Capture ELISA can accurately measure the affinity of antibodies to Aβ monomers. 96-well plates were coated with 5 μg / mL of antibody overnight at room temperature. Biotin-labeled Aβ was then added. 1-40 Plates were treated with dilutions of monomer (AnaSpec, AS-61483-01, 4-fold dilution series with concentrations ranging from 0 to 1000 nM) for 1 h at RT. Biotin-labeled Aβ was then incubated with immobilized antibody as described in Example 2. 1-40 Monomer binding was detected by HRP-conjugated streptavidin and TMB. The EC50 of the antibody for Aβ monomer (monomer EC50) was determined in this assay (Figure 7 and Table 1). The ratio of the monomer EC50 in the capture ELISA to the oligomer IC50 in the competitive ELISA was then calculated. This ratio reflects the selectivity of the antibody for Aβ protofibrils over Aβ monomers and was used to rank the antibodies as shown in Table 1.

[0107] Antibody-dependent cell-mediated phagocytosis (ADCP)

[0108] Following the non-limiting hypothesis that anti-Aβ antibodies can clear toxic Aβ protofibrils / plaques in the brain by inducing microglia to ADCP of Aβ, the ability of lead antibodies to mediate the phagocytosis of Aβ protofibrils by microglia was assessed using HiLyte488-labeled Aβ protofibrils and the mouse microglial cell line BV-2 cells. 1-42 Monomers were labeled with unlabeled Aβ 1-42The oligomers were mixed with the monomer at a 1:5 ratio and incubated at 37°C for 2 hours to form HiLyte488-labeled protofibrils, which were then purified by SEC separation and protofibril peak collection. BV-2 cells were seeded at a density of 60,000 cells per well in a 96-well cell culture plate and incubated overnight. The BV-2 cells were then pretreated with ADCP assay diluent (DMEM / F12 containing HEPES medium, 1% BSA, and 100 μg / mL fucoidan) for 1 hour prior to the ADCP assay. Next, 2.25 μg / mL of HiLyte488-labeled Aβ oligomers were mixed with a dilution series of each lead antibody or mouse IgG2b (negative control) in ADCP assay diluent (800 ng / mL, 160 ng / mL, 32 ng / mL, and 6.4 ng / mL) for 30 minutes. The mixture of HiLyte488-labeled Aβ protofibrils and antibodies was added to BV-2 cells and incubated at 37°C for 1.5 hours to induce ADCP. Cell surface-bound oligomer-antibody complexes were removed by treatment with 0.25% trypsin for 20 minutes at 4°C. Cells were transferred to a conical-bottom 96-well plate, rinsed twice with ice-cold cell staining buffer (BioLegend #420201), fixed for 20 minutes with ice-cold fixation buffer (BioLegend #420801), and washed once again. Finally, cells were analyzed by flow cytometry for a positive FITC signal, reflecting the amount of HiLyte488-labeled Aβ protofibrils internalized by BV-2 cells (Figure 8). The ADCP response was calculated using raw RFU (relative fluorescence unit) values ​​(Table 1), and the ability of each lead antibody candidate to elicit ADCP could be assessed relative to the ADCP response of rfmAb-1. Antibodies 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A had relative ADCPs of 1.36, 0.79, 1.05, 1.39, and 0.95, respectively.

[0109] Example 5: Reformatting and Recombinant Expression

[0110] Reformatting as a fully human antibody

[0111] As described in Example 1, five lead antibodies, 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A, were originally isolated from hybridoma supernatants, subcloned, and purified to generate chimeric monoclonal antibodies having human F(ab')2 and mouse IgG1 CH2 and CH3 domains (i.e., the chimeric human F(ab')2 / mouse IgG1 antibodies 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 lead antibodies can be identified as 18P01A (mIgG1), 17P04A (mIgG1), 20O07A (mIgG1), 22D04A (mIgG1), and 06E17A (mIgG1), as shown in Tables 4 and 5.

[0112] Each of the five original chimeric human F(ab')2 / mouse IgG1 lead antibodies was then reformatted into fully human IgG1 lambda antibodies by replacing the heavy chain constant region of each antibody with a human IgG1 heavy chain constant region, including CH1, hinge region, CH2, and CH3, via de novo gene synthesis, such that the heavy chain (HC) amino acid sequence of each reformatted fully human antibody comprises the VH amino acid sequence (including HC CDRs 1-3) of the source mAb, and the light chain (LC) amino acid sequence of each reformatted fully human comprises the VL amino acid sequence (LC CDRs 1-3) of the source mAb.

[0113] Fully human antibody 18P01A (hIgG1) has an HC amino acid sequence of SEQ ID NO: 157, which comprises the 18P01A VH amino acid sequence of SEQ ID NO: 61, and an LC amino acid sequence of SEQ ID NO: 158, which comprises the 18P01A VL amino acid sequence of SEQ ID NO: 66. Fully human antibody 17P04A (hIgG1) has an HC amino acid sequence of SEQ ID NO: 153, which comprises the 17P04A VH amino acid sequence of SEQ ID NO: 41, and an LC amino acid sequence of SEQ ID NO: 154, which comprises the 17P04A VL amino acid sequence of SEQ ID NO: 46. Fully human antibody 20O07A (hIgG1) has an HC amino acid sequence of SEQ ID NO: 161, which comprises the 20O07A VH amino acid sequence of SEQ ID NO: 71, and an LC amino acid sequence of SEQ ID NO: 162, which comprises the 20O07A VL amino acid sequence of SEQ ID NO: 76. Fully human antibody 22D04A (hIgG1) has an HC amino acid sequence of SEQ ID NO: 165, which comprises the 22D04A VH amino acid sequence of SEQ ID NO: 131, and an LC amino acid sequence of SEQ ID NO: 166, which comprises the 22D04A VL amino acid sequence of SEQ ID NO: 136. Fully human antibody 06E17A (hIgG1) has an HC amino acid sequence of SEQ ID NO: 169, which comprises the 06E17A VH amino acid sequence of SEQ ID NO: 1, and an LC amino acid sequence of SEQ ID NO: 169, which comprises the 06E17A VL amino acid sequence of SEQ ID NO: 6.

[0114] The reformatted fully human antibodies were expressed in CHO cells via transient transfection using the ExpiCHO expression system and purified using a Protein A column. The purified fully human antibodies were 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 with human F(ab')2 / mouse IgG1 CH2-CH3. The results are shown in Table 4.

[0115] For antibodies 17P04A, 20O07A, 22D04A, and 06E17A, there was no difference in the profiles between the fully human versions and the original chimeric human F(ab')2 / mouse IgG1 versions. However, fully human 18P01A showed a dramatic reduction in affinity for Aβ in all three conformations tested by competitive ELISA. Results from direct ELISA showed that fully human 18P01A had significantly reduced affinity for Aβ. 1-42 EC 50 was shown to be increased by >800-fold to 49.96 nM compared to 0.062 mM for the original chimeric 18P01A.

[0116] Table 4. Comparison of fully human and mouse IgG1 / human chimeric versions of anti-Aβ antibodies. UND indicates undetermined value.

[0117] [Table 4]

[0118] Reformatting as human F(ab')2 / mouse IgG2a CH2-CH3 chimeric antibody

[0119] Conversion of mAbs from mouse IgG1 to mouse IgG2a may enhance their ADCP activity. To test their effect on ADCP, the original lead antibodies 18P01A, 17P04A, 20O07A, 22D04A, and 06E17A were reformatted from the original mouse IgG1 / human chimeric version (mouse IgG1 / human F(ab')2) to a mouse IgG2a / human chimeric version (mouse IgG2a CH2-CH3 / human F(ab')2) for animal testing. The reformatted antibody 18P01A (mIgG2a) has a heavy chain (HC) amino acid sequence of SEQ ID NO: 155, which contains the 18P01A VH amino acid sequence of SEQ ID NO: 61, and a LC amino acid sequence of SEQ ID NO: 156, which contains the 18P01A VL amino acid sequence of SEQ ID NO: 66. Reformatted antibody 17P04A (mIgG2a) has an HC amino acid sequence of SEQ ID NO: 151, which comprises the 17P04A VH amino acid sequence of SEQ ID NO: 41, and an LC amino acid sequence of SEQ ID NO: 152, which comprises the 17P04A VL amino acid sequence of SEQ ID NO: 46. Reformatted antibody 20O07A (mIgG2a) has an HC amino acid sequence of SEQ ID NO: 159, which comprises the 20O07A VH amino acid sequence of SEQ ID NO: 71, and an LC amino acid sequence of SEQ ID NO: 160, which comprises the 20O07A VL amino acid sequence of SEQ ID NO: 76. Reformatted antibody 22D04A (mIgG2a) has an HC amino acid sequence of SEQ ID NO: 163, which comprises the 22D04A VH amino acid sequence of SEQ ID NO: 131, and an LC amino acid sequence of SEQ ID NO: 164, which comprises the 22D04A VL amino acid sequence of SEQ ID NO: 136. The reformatted antibody 06E17A (mIgG2a) has an HC amino acid sequence of SEQ ID NO: 167, which comprises the 06E17A VH amino acid sequence of SEQ ID NO: 1, and an LC amino acid sequence of SEQ ID NO: 168, which comprises the 06E17A VL amino acid sequence of SEQ ID NO: 6.

[0120] The 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 version 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).

[0121] Table 5. Comparison of the original human F(ab')2 / mouse IgG1 chimeric and reformatted human F(ab')2 / mouse IgG2a CH2-CH3 versions of 17P04A and 18P01A. UND is an undetermined value. [Table 5]

[0122] Example 6: Pharmacokinetics of 17P04A and 18P01A in mice

[0123] The human F(ab')2 / mouse IgG2a CH2-CH3 chimeric versions, designated 17P04A (mIgG2a) and 18P01A (mIgG2a), were selected for pharmacokinetic studies based on their high level of selective binding to Aβ protofibrils and their ability to induce ADCP. Antibodies 17P04A (mIgG2a) and 18P01A (mIgG2a) were injected intraperitoneally (ip) at a dose of 10 mg / kg into B6SJLF1 mice (Jackson Laboratory, JAX#100012, jax.org; MPD ID:178, phenome.jax.org / strains / 178). For use as a control, rfmAb-1 was also administered i.p. to a group of B6SJLF1 mice (Jackson Laboratory, JAX#100012) at the same dose as rfmAb01 (mIgG2b). Serum and brain tissue were collected 4 hours, 8 hours, 1 day, 2 days, 3 days, 7 days, 10 days, and 14 days after injection. Before collecting brain tissue, animals were whole-body perfused with PBS. Brain tissue was homogenized in a 3x volume (300 μL per 100 mg of tissue) of TBS containing a protease inhibitor cocktail (Thermo Fisher Scientific, 78429; thermofisher.com) using a Bullet Blender 5E Gold (NextAdvance, BB5E-AU) with a zirconium oxide bed (NextAdvance, PINK5E100). The homogenate was transferred to a 2 mL tube and centrifuged at 16,000 × g for 20 min at 4 °C to remove the pellet. The whole blood was allowed to clot at room temperature for 30 min, then centrifuged and the separated serum was collected. Drug concentrations in the supernatant of the brain homogenate and serum were measured using the ELISA kit. 1-42 The plate was coated with 0.5 μM Aβ peptide and measured by direct ELISA. 1-42Plates were coated with rPeptide (A-1167-2, rPeptide.com) overnight at 4°C, washed three times with TBST, and blocked with protein-free blocking buffer (ThermoFisher, 37572) for 1 hour at RT. Dilutions of brain homogenate (2-fold dilutions starting at 1:4, triplicates per concentration) or serum (2-fold dilutions starting at 1:200, triplicates per concentration) were applied to the plate for 1 hour at RT. Immobilization of antibodies in brain homogenate or serum 1-42 Binding to rfmAb-1 was detected using an HRP-conjugated secondary antibody and TMB substrate as described in the direct ELISA protocol in Example 2. Non-compartmental pharmacokinetic (PK) analysis was performed via an online PK calculator (non-compartmental pharmacokinetic analysis calculator available at dash.gallery / dash-pk-calc / ). The half-lives of rfmAb-1 (mIgG2a), 17P04A (mIgG2a), and 18P01A (mIgG2a) in serum 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 brain were 28.2 hours, 92.4 hours, and 77.2 hours, respectively (Figure 10). Brain penetration, calculated as the ratio of brain AUC (area under the curve) to serum AUC, was 1.9% for rfmAb-1 (mIgG2a), 5.5% for 17P04A (mIgG2a), and 0.3% for 18P01A (mIgG2a) (Figure 10). Similarly, 17P04A (mIgG2a) had a longer half-life in serum and brain and better brain penetration than 18P01A (mIgG2a) or rfmAb-1 (mIgG2a). Despite its longer half-life in serum and brain, 18P01A (mIgG2a) exhibited poor brain penetration, which may limit its efficacy in clearing Aβ in the brains of AD patients or in AD mouse models.

[0124] Example 7: Long-term efficacy study of 17P04A in 5xFAD mice

[0125] 5xFAD transgenic mice overexpressed mutant human amyloid beta precursor protein (APP) harboring the Swedish (K670N, M671L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) mutations, as well as human presenilin 1 (PSEN1) harboring two FAD mutations, M146L and L286V. These mice developed numerous AD-related phenotypes at a relatively early age. Here, we used 5xFAD mice as an AD mouse model to evaluate and compare the long-term efficacy of an antibody containing the 17P04A variable region (17P04AFab), which is highly selective for Aβ oligomers / protofibrils, and rfmAb-1 (see above). Seven-month-old 5xFAD mice (Jackson Laboratory, JAX#034840, jax.org, RRID:MMRRC_034840-JAX) on a B6SJLF1 / J background were injected weekly with 1, 3, and 10 mg / kg of 17P04A (mIgG2a) or 1, 3, and 10 mg / kg of rfmAb-1 (mIgG2a) i.p. for 16 weeks. 5xFAD mice received weekly i.p. injections of 1x PBS (vehicle) and age-matched naive B6SJLF1 / J mice (wild-type) served as controls. Each treatment group contained 10–15 mice.

[0126] Aβ in brain homogenate fractions 1-42 level, Aβ 1-40 Measurement of Aβ levels, and Aβ oligomer / protofibril levels

[0127] Three days after the last injection, mice were anesthetized with 250 mg / kg tribromoethanol and transcardially perfused with approximately 15 mL of 1x PBS (pH 7.4). Half of the cerebellum from each PBS-perfused mouse was homogenized in 600 μL of 1x Tris-buffered saline (TBS) (pH 7.4) containing a protease / phosphatase inhibitor cocktail (Thermo Scientific, 78444) using a Bullet Blender (NextAdvance, BB5E-AU). 250 μL of the homogenate was centrifuged at 20,000 × g for 20 minutes at 4°C. The supernatant was designated the TBS-soluble fraction. The remaining 250 μL of homogenate was added to an equal volume of RIPA buffer (Thermo Scientific, 89901) and centrifuged at 20,000 × g for 20 minutes at 4°C. The supernatant was used as the RIPA-soluble fraction. The pellet was resuspended in 100 μL of 8 M guanidine-HCl and incubated at room temperature for 2 hours. The resuspension was diluted by adding 400 μL of 1× TBS and centrifuged again at 20,000 × g for 20 minutes at 4°C. The supernatant was used as the guanidine-soluble fraction.

[0128] Aβ in the RIPA-soluble and guanidine-soluble fractions was assayed using the Human Amyloid β (aa1–42) Quantikine ELISA Kit (R&D Systems, DAB142, rndsystems.com) according to the manufacturer's instructions. 1-42 The levels of Aβ in the RIPA soluble fraction (mostly Aβ monomers) were measured. The results are shown in Figures 11A and 11B. As shown in Figure 11A, the Aβ levels in the RIPA soluble fraction (mostly Aβ monomers) were 1-42 The levels of Aβ in the guanidine-soluble fraction, i.e., the fraction containing mostly insoluble Aβ fibrils and plaques, were not affected by 17P04A (mIgG2a) or rfmAb-1 at any dose. 1-42 Figure 11B shows that Aβ levels were significantly reduced by injection of 1, 3, and 10 mg / kg of 17P04A (mIgG2a) and by injection of 3 and 10 mg / kg of mAb158. Importantly, Figure 11B shows that Aβ levels in mice treated with 17P04 (mIgG2a) were significantly reduced by injection of 1, 3, and 10 mg / kg of mAb158. 1-42It was shown that the levels were significantly lower than those in mice treated with the same dose of rfmAb-1 (p<0.05 for 3 mg / kg rfmAb-1 vs. 3 mg / kg 17P04A(mIgG2a); p<0.01 for 10 mg / kg rfmAb-1 vs. 10 mg / kg 17P04A(mIgG2a)), suggesting that 17P04A(mIgG2a) clears Aβ plaques more effectively than the reference antibody rfmAb-1.

[0129] Aβ 1-40 is an abundant Aβ isoform in the brain. It is considered non-toxic due to its lack of tendency to aggregate. Aβ in the RIPA-soluble and guanidine-soluble fractions was assayed using the Human Amyloid β (aa1-40) Quantikine ELISA Kit (R&D Systems, DAB140, rndsystems.com) according to the manufacturer's instructions. 1-40 The levels of Aβ were measured in both the RIPA-soluble fraction (FIG. 12A) and the guanidine-soluble fraction (FIG. 12B) of each sample. 1-40 Levels were not affected by weekly ip injections of 1 mg / kg, 3 mg / kg, or 10 mg / kg of 17P04 (mIgG2a) or 3 mg / kg or 10 mg / kg of rfmAb-1. Treatment with 1 mg / kg of rfmAb-1 reduced Aβ levels in the RIPA soluble fraction for unknown reasons. 1-40 The level has increased significantly.

[0130] Aβ oligomer / protofibril levels in the TBS-soluble fraction of brain were measured using a sandwich ELISA using rfmAb-1 as the capture antibody and biotin-labeled 18P01 as the detection antibody. The results are shown in Figure 13. For the assay, 96-well plates were coated with 100 μL per well of 2 μg / mL rfmAb-1 in PBS overnight at 4°C. The plates were blocked with 1% BSA in PBS for 1 hour at RT. Then, 100 μL per well of the TBS-soluble fraction (diluted 1 / 40) or purified Aβ was added. 1-42Oligomers (2-fold dilutions with concentrations ranging from 0 to 2000 pg / mL) were added to the plates. The plates were incubated at RT for 1 h and washed three times. Plates were incubated with 100 μL per well of 1 μg / mL biotinylated 18P01 in 1% BSA for 1 h and washed three times. Plates were then incubated with 100 μL per well of HRP-conjugated streptavidin (Molecular Probes, SNN2004) diluted 1:5000 in 1% BSA for 1 h and washed three times. TMB Stabilized Chromogen substrate solution was added to the plates, and the reaction was stopped by adding 1 M sulfuric acid. Absorbance at 450 nm was measured in a plate reader. Aβ oligomer levels in WT mice were considered background signals, and values ​​were subtracted from all groups. The results showed that treatment with 17P04 dose-dependently reduced Aβ oligomers in the brain (5%, 34%, and 68% reductions at 1, 3, and 10 mg / kg, respectively) (Figure 13). Treatment with 10 mg / kg of 17P04A(mIgG2a) reduced Aβ oligomer / protofibril levels significantly lower than vehicle treatment (Veh), i.e., the control. Treatment with 10 mg / kg of rfmAb-1 reduced Aβ oligomers by 36% compared to the vehicle-treated group (control), but the difference was not significant. These results indicated that 17P04A(mIgG2a) reduced Aβ oligomers / protofibrils in the brain. These results suggest that 17P04A(mIgG2a) reduces Aβ oligomers / protofibrils in the brain more effectively than rfmAb-1.

[0131] Aβ 1-40 and Aβ 1-42 Aβ is present in serum mostly as a monomer. Aβ in the serum of treated mice was measured using the LEGEND MAX™ Human Amyloid Beta (1-42) ELISA Kit (BioLegend, 448707) according to the kit's instructions. 1-42 The levels were measured (Figure 14). 1-42Aβ levels were unchanged in the serum of mice treated with 1 mg / kg or 3 mg / kg of 17P04A (mIgG2a) or 1 mg / kg, 3 mg / kg, or 10 mg / kg of rfmAb-1. 1-42 Levels were significantly increased in the serum of mice treated with 10 mg / kg of 17P04A (mIgG2a) for unknown reasons. Aβ levels in the serum of treated mice were measured using the LEGEND MAX™ Human Amyloid Beta (1-40) ELISA Kit (BioLegend, 449007). 1-40 The levels of Aβ in serum were measured (Figure 14). 1-40 Levels were not affected by treatment with rfmAb01 or 17P04A (mIgG2a) at any dose.

[0132] Measurement of plaques in fixed brain tissue

[0133] The other half of the cerebrum from each PBS-perfused mouse was fixed in 4% PFA in PBS for 1 day, incubated in 1x PBS for 1 day, and then sent to NeuroScience Associates (neuroscienceassociates.com) for Campbell-Switzer Alzheimer's staining to reveal Aβ plaques. Twenty-five brain hemispheres were embedded together in a single block and cryosectioned at 35 μm in the coronal plane across the cortical compartment of each mouse brain hemisphere. Campbell-Switzer staining was performed across the cerebrum on every sixth section spaced 210 μm apart. Slides (25 brain sections per slide) were washed three times with dH2O, placed in 2% NH4OH for 5 minutes, and washed once more with dH2O. Slides were then placed in silver-pyridine-carboxylic acid 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 Associates) and then developed in fresh physical developer ABS solution (Neuroscience Associates). Development times were matched between slides. Development was stopped by briefly placing the slides in 4.99 pH acetate buffer working solution. Slides were washed in dH2O for 30 seconds and placed in 0.5% sodium thiosulfate solution for 45 seconds. After a final wash in dH2O (3 × 2 min), the slides were coverslipped. Slides were then imaged under a bright-field microscope. Representative images of Campbell-Switzer staining were shown in Figure 15 using ImageJ software (imagej.net) for image processing and analysis. The area percentages of dense and diffuse plaques in each brain section were quantified. The cortical and hippocampal areas of each brain section were outlined, and the total area was measured. To measure dense plaques, the image threshold was adjusted to 0, 20. To measure dense and diffuse plaques, the image threshold was adjusted to 0 and 50. Plaque area was measured using the particle analysis function (Analyze Particle) in ImageJ. Particle size was set to 10 to infinity. Area percentage was calculated by dividing the plaque area by the total area.The area percentages of four brain sections at bregma -0.25 mm, -1.30 mm, -2.35 mm, and -3.40 mm were measured and averaged. Eight to thirteen brains were analyzed for each treatment group. The results in Figure 15 demonstrate that treatment of 5xFAD mice with 3 mg / kg and 10 mg / kg of 17P04A (mIgG2a) significantly reduced dense and diffuse plaques in the brains of 5xFAD mice. Treatment with rfmAb-1 at any dose did not significantly affect the levels of dense or diffuse plaques.

[0134] Results from a long-term efficacy study showed that treatment with 17P04A(mIgG2a) resulted in a statistically significant reduction in soluble Aβ oligomers / protofibrils in the brains of 5×FAD mice compared with control 5×FAD mice treated with vehicle (PBS) for the same time period ( FIG. 13 ), suggesting that 17P04A(mIgG2a) effectively clears soluble Aβ oligomers / protofibrils in the brains of an in vivo AD disease model.

[0135] Results from a long-term efficacy study showed that treatment with 17P04A(mIgG2a) resulted in a statistically significant reduction in dense and insoluble plaques in the brains of 5×FAD mice compared with control 5×FAD mice treated with vehicle (PBS) for the same time period ( FIG. 15 ). These results indicate that 17P04A(mIgG2a) reduced dense and diffuse plaques in the brains of 5×FAD mice more effectively than the reference antibody rfmAb-1. This suggests that 17P04A(mIgG2a) reduces both dense and insoluble Aβ fibrils / plaques in the brains of in vivo AD disease models. These results demonstrate that treatment with 17P04A(mIgG2a) can effectively remove and / or reduce brain Aβ plaques, thereby lowering plaque burden, in animals affected by a condition associated with at least one Aβ protein aggregation, specifically AD.

Claims

1. an anti-Aβ protofibril / oligomer antibody and its antibody fragment that preferentially binds to soluble Aβ protofibrils / oligomers, wherein a complex comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to Aβ protofibrils / oligomers can induce antibody-dependent cell-mediated phagocytosis (ADCP) by microglial cells after exposure of the complex to the microglial cells, and the anti-Aβ protofibril / oligomer antibody or antibody fragment has a fully human variable region; (a) an antibody or antibody fragment comprising a fully human heavy chain variable region (VH) comprising an HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO: 42), an HC CDR2 having the amino acid sequence ISSRRTYI (SEQ ID NO: 43), and an HC CDR3 having the amino acid sequence ARGGYIGSPNAYDI (SEQ ID NO: 44), and a fully human light chain variable region (VL) comprising 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 fully human heavy chain variable region (VH) comprising 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 TSHAPNFDAFDI (SEQ ID NO: 64), and a fully human light chain variable region (VL) comprising an LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 67), an LC CDR2 having the amino acid sequence DNS, and an LC CDR3 having the amino acid sequence GTWDSSLSTYV (SEQ ID NO: 69); (c) an antibody or antibody fragment comprising a fully human heavy chain variable region (VH) comprising an HC CDR1 having the amino acid sequence GFTFSGSA (SEQ ID NO: 72), an HC CDR2 having the amino acid sequence IRSKVNSYAT (SEQ ID NO: 73), and an HC CDR3 having the amino acid sequence TSHAP IFDAFDI (SEQ ID NO: 74), and a fully human light chain variable region (VL) comprising an LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 77), an LC CDR2 having the amino acid sequence DNS, and an LC CDR3 having the amino acid sequence GTWDSSLSTYF (SEQ ID NO: 79); (d) an antibody or antibody fragment comprising a fully human heavy chain variable region (VH) comprising an HC CDR1 having the amino acid sequence GFTFSNAW (SEQ ID NO: 132), an HC CDR2 having the amino acid sequence IKSKTDGGTR (SEQ ID NO: 133), and an HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 134), and a fully human light chain variable region (VL) comprising an LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO: 137), an LC CDR2 having the amino acid sequence RNN, and an LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 139); and (e) an antibody or antibody fragment comprising a fully human heavy chain variable region (VH) comprising an HC CDR1 having the amino acid sequence GFSFSNAW (SEQ ID NO: 2), an HC CDR2 having the amino acid sequence IKSKTDGGTI (SEQ ID NO: 3), and an HC CDR3 having the amino acid sequence TTGYGEGY (SEQ ID NO: 4), and a fully human light chain variable region (VL) comprising an LC CDR1 having the amino acid sequence SSNIKSNT (SEQ ID NO: 7), an LC CDR2 having the amino acid sequence RNN, and an LC CDR3 having the amino acid sequence AAWDDSLKGVV (SEQ ID NO: 9); An antibody or antibody fragment selected from one of:

2. an antibody or antibody fragment comprising 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; an antibody or antibody fragment comprising a fully human heavy chain variable region VH having the amino acid sequence of SEQ ID NO: 61 and a fully human light chain variable region VL having the amino acid sequence of SEQ ID NO: 66; an antibody or antibody fragment comprising a fully human heavy chain variable region VH having the amino acid sequence of SEQ ID NO: 71 and a fully human light chain variable region VL having the amino acid sequence of SEQ ID NO: 76; An antibody or antibody fragment comprising a fully human heavy chain variable region VH having the amino acid sequence of SEQ ID NO: 131 and a fully human light chain variable region VL having the amino acid sequence of SEQ ID NO: 136; and An antibody or antibody fragment 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. The anti-Aβ oligomer antibody or antibody fragment according to claim 1 , selected from one of the following:

3. 3. The anti-Aβ protofibril / oligomer antibody or antibody fragment of claim 1, which is capable of penetrating brain tissue of a mammalian subject after administration.

4. an antibody or antibody fragment comprising: a fully human heavy chain variable region (VH) comprising an HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO: 42), an HC CDR2 having the amino acid sequence ISSRRTYI (SEQ ID NO: 43), and an HC CDR3 having the amino acid sequence ARGGYIGSPNAYDI (SEQ ID NO: 44); and a fully human light chain variable region (VL) comprising 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); an antibody or antibody fragment comprising 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; an antibody or antibody fragment comprising a fully human heavy chain variable region (VH) comprising 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 TSHAPNFDAFDI (SEQ ID NO: 64); and a fully human light chain variable region (VL) comprising an LC CDR1 having the amino acid sequence SSNIGNHY (SEQ ID NO: 67), an LC CDR2 having the amino acid sequence DNS, and an LC CDR3 having the amino acid sequence GTWDSSLSTYV (SEQ ID NO: 69); and an antibody or antibody fragment comprising a fully human heavy chain variable region VH having the amino acid sequence of SEQ ID NO: 61 and a fully human light chain variable region VL having the amino acid sequence of SEQ ID NO: 66; The anti-Aβ oligomer antibody or antibody fragment according to claim 3 , wherein the antibody or antibody fragment is selected from one of the following:

5. 4. The anti-Aβ protofibril / oligomer antibody or antibody fragment of claim 3, which is capable of reducing the level of soluble Aβ oligomers / protofibrils in the brain of the mammalian subject after administration.

6. 4. The anti-Aβ protofibril / oligomer antibody or antibody fragment of claim 3, which is capable of reducing the level of Aβ plaques in the brain of the mammalian subject after administration.

7. 7. The anti-Aβ protofibril / oligomer antibody or antibody fragment of claim 1 , wherein the constant region sequence involved in eliciting ADCP comprises a sequence derived from an IgG fragment crystallizable region (Fc), optionally selected from mouse IgG1 Fc, human IgG1 Fc, and mouse IgG2a Fc.

8. A pharmaceutical composition comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment of any one of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient.

9. 9. An anti-Aβ protofibril / oligomer antibody or antibody fragment according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in reducing the amount of soluble Aβ protofibrils / oligomers in a subject after administration to the subject.

10. Use of an anti-Aβ protofibril / oligomer antibody or antibody fragment described in any one of claims 1 to 7 or a pharmaceutical composition described in claims 8 and 9 for reducing the amount of soluble Aβ protofibrils / oligomers in at least one of the blood, lymph, cerebrospinal fluid (CSF), nervous tissue, and brain tissue of a subject after administration.

11. Use of the anti-Aβ protofibril / oligomer antibody or antibody fragment of any of claims 1 to 7 or the pharmaceutical composition of claims 8 or 10, for binding to soluble Aβ protofibrils / oligomers in at least one of the blood, lymph, cerebrospinal fluid (CSF), neural tissue, and brain tissue of the subject after administration, and inducing ADCP of a complex comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment bound to the Aβ protofibril / oligomer.

12. Use of an anti-Aβ protofibril / oligomer antibody or antibody fragment described in any one of claims 1 to 7 or a pharmaceutical composition described in claims 8 and 10 for reducing the amount of soluble Aβ protofibrils / oligomers in the brain tissue of the subject.

13. The use of claim 12, wherein the anti-Aβ oligomer antibody or antibody fragment comprises a fully human VH comprising a HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO: 42), a HC CDR2 having the amino acid sequence ISSRRTYI (SEQ ID NO: 43), and a HC CDR3 having the amino acid sequence ARGGYIGSPNAYDI (SEQ ID NO: 44), and a fully human VL comprising a LC CDR1 having the amino acid sequence TGAVTSDYY (SEQ ID NO: 47), a LC CDR2 having the amino acid sequence SAS, and a LC CDR3 having the amino acid sequence LLYYGGAWV (SEQ ID NO: 49).

14. The use of claim 12, wherein the anti-Aβ oligomer antibody or antibody fragment comprises 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.

15. 9. An anti-Aβ protofibril / oligomer antibody or antibody fragment according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8, for use in reducing the amount of Aβ plaques in the brain of a subject after administration.

16. The use of claim 15, wherein the anti-Aβ oligomer antibody or antibody fragment comprises a fully human VH comprising a HC CDR1 having the amino acid sequence GFTLSSFS (SEQ ID NO: 42), a HC CDR2 having the amino acid sequence ISSRRTYI (SEQ ID NO: 43), and a HC CDR3 having the amino acid sequence ARGGYIGSPNAYDI (SEQ ID NO: 44), and a fully human VL comprising a LC CDR1 having the amino acid sequence TGAVTSDYY (SEQ ID NO: 47), a LC CDR2 having the amino acid sequence SAS, and a LC CDR3 having the amino acid sequence LLYYGGAWV (SEQ ID NO: 49).

17. The use of claim 15, wherein the anti-Aβ oligomer antibody or antibody fragment comprises 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.

18. A composition comprising an anti-Aβ protofibril / oligomer antibody or antibody fragment according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8, for use in treating at least one condition associated with aggregation of Aβ protein.

19. 19. The composition of claim 18 for use in preventing or delaying the development of at least one condition associated with aggregation of Aβ protein.

20. 20. The composition of claim 18 or 19, wherein the condition associated with Aβ protein aggregation is Alzheimer's disease (AD).

21. 10. A method for detecting soluble Aβ protofibrils and / or oligomers in a sample, the method comprising: contacting the sample with a composition comprising the anti-Aβ protofibril / oligomer antibody or antibody fragment of any one of claims 1 to 7; and detecting binding of the anti-Aβ protofibril / oligomer antibody or antibody fragment to Aβ protofibrils and / or oligomers, thereby indicating the presence of soluble Aβ protofibrils and / or oligomers in the sample.

22. 22. The method of claim 21, further comprising determining the amount of soluble Aβ protofibrils and / or oligomers in the sample.

23. 3. An isolated nucleic acid molecule encoding at least a portion of the anti-Aβ protofibril / oligomer antibody or antibody fragment of claim 2, comprising a nucleotide sequence that is one of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:75, SEQ ID NO:80, SEQ ID NO:135, and SEQ ID NO:140.

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