Methods and materials for treating neurodegenerative diseases

Abstract

Described herein are methods for producing and using antibodies that specifically bind to soluble multimers of amyloid beta protein (oAβ), and for using them in the diagnosis and treatment of diseases associated with the formation of pathogenic multimers of oAβ.Also provided are helper T cell (Th) carrier peptides that can be conjugated to peptide immunogens to stimulate targeted humoral immune responses against desired antigens.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Application No. 63 / 238,252, filed August 30, 2021, U.S. Provisional Application No. 63 / 238,329, filed August 30, 2021, U.S. Provisional Application No. 63 / 346,240, filed May 26, 2022, and U.S. Provisional Application No. 63 / 393,885, filed July 30, 2022. The entire contents of the above references are incorporated herein by reference.

[0002] Federally sponsored research or development This invention was made with government support under Grant Nos. AG006173, AG015379, AG071865, and AG063046 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Described herein are compositions and methods for making and using antibodies that specifically bind to soluble multimers of amyloid beta protein (oAβ), and methods for using them in the diagnosis and treatment of diseases associated with the formation of pathogenic multimers of oAβ.Also provided are helper T cell (Th) carrier peptides that can be conjugated to peptide immunogens to stimulate targeted humoral immune responses against desired antigens. [Background technology]

[0004] Alzheimer's disease (AD) is a slowly progressive neurodegenerative disease at both histopathological and clinical levels, responsible for approximately 70% of all dementias. Initial symptoms of temporary mild memory loss and minimal intermittent cognitive impairment gradually lead to severe dementia and death over 5–15 years. Currently, an estimated 6.2 million Americans and over 45 million people worldwide live with Alzheimer's disease, causing significant family, social, and economic burdens. AD is the fifth leading cause of death among older Americans, and in 2020, unpaid caregiving for patients with dementia was valued at $256.7 billion (2021 Alzheimer's facts and figures). Until the approval of aducanumab in June 2021, only five drugs were approved for the treatment of AD: four cholinesterase inhibitors and one N-methyl-D-aspartate (NMDA) receptor antagonist, which are only symptomatic and have minimal efficacy, and none of these drugs treat the underlying cause of AD or slow cognitive decline (Cummings et al., 2016). Aducanumab, sold under the trade name Aduherm, is the first disease-modifying therapy available for the treatment of Alzheimer's disease. Aduherm has been shown to have high specificity for insoluble Aβ aggregates and reduce brain Aβ plaque burden in a time- and dose-dependent manner (Arndt et al., 2018;Sevigny et al., 2016). Although this approval provides important clinical support for the amyloid hypothesis and demonstrates Aβ as a drug target, serious doubts remain about the drug's efficacy on cognitive decline, despite Aduherm's positive target engagement and ability to reduce plaque burden (Knopman et al., 2021). Aduherm's approval is a boon for patients and families suffering from AD and highlights the urgent need to develop more effective and affordable AD diagnostic strategies and disease-modifying therapies. Summary of the Invention

[0005] Described herein are compositions and methods for making and using antibodies that specifically bind to soluble multimers of amyloid beta protein (oAβ), and methods for using them in the diagnosis and treatment of diseases associated with the formation of pathogenic multimers of oAβ.Also provided are helper T cell (Th) carrier peptides that can be conjugated to peptide immunogens to stimulate targeted humoral immune responses against desired antigens.

[0006] Thus, provided herein is a synthetic peptide immunogen for stimulating antibody generation, the synthetic peptide immunogen being specific for a conformational epitope present in aggregated forms of amyloid beta protein (Aβ), including multimers of amyloid beta protein (oAβ), but not specific for the monomeric form. In some embodiments, the synthetic peptide immunogen comprises the sequence GYEVHHQKLV (SEQ ID NO: 1) or the sequence YEXHH (SEQ ID NO: 41), where X is a hydrophobic amino acid (V, I, or L).

[0007] In some embodiments, the synthetic peptide immunogen comprises the sequence CGKCGYEVHHQKLVPNVLKQHHVEYGCK (SEQ ID NO: 2) or CGKCGYEVHHQKLVPNGYEVHHQKLVCK ​​(SEQ ID NO: 3). In some embodiments, the synthetic peptide immunogen comprises the sequence shown in Figure 13, i.e., SEQ ID NOs: 5-17. In some embodiments, the synthetic peptide immunogen comprises the sequence of peptides 1, 3, 5, 7, 8, 11, or 13, with peptides 5, 8, or 7 being most preferred.

[0008] In some embodiments, the synthetic peptide immunogen further comprises a carrier protein linked to the N-terminus, optionally the carrier protein is linked by a maleimide, hi some embodiments, the synthetic peptide immunogen comprises a carrier protein comprising keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or ovalbumin (OVA).

[0009] In some embodiments, the synthetic peptide immunogen optionally comprises (i) a carrier protein comprising a peptide having Pam3Cys (N-α-palmitoyl-S-2,3-bis(palmitoyloxy)-(2RS)-propyl-L-cysteine, where palmitoyl-Cys ((RS)-2,3) is attached to the second lysine (K4), the sequence KSSKSKKKFISEAIIHHLHSRHPGK (SEQ ID NO: 4), wherein the carrier protein is conjugated to the N-terminus via a maleimide.

[0010] Also provided herein are synthetic peptides, optionally having (i) Pam3Cys (N-α-palmitoyl-S-2,3-bis(palmitoyloxy)-(2RS)-propyl-L-cysteine), with the sequence KSSKSKKKFISEAIIHHLHSRHPGK (SEQ ID NO: 4) in which palmitoyl-Cys ((RS)-2,3) is attached to the second lysine (K4), and methods for their use to increase an immune response to an antigen.

[0011] Additionally, compositions comprising the peptides described herein are provided.

[0012] Additionally, provided herein is a method for detecting oAβ in a sample comprising one or more biological fluids, preferably in a sample derived from a human subject. Such a method includes contacting the sample with a capture antibody bound to a surface, the capture antibody comprising 71A1 antibody or 1G5 antibody, under conditions sufficient for the formation of a capture antibody-oAβ complex, and optionally washing to remove unbound protein; contacting the sample with a labeled detection antibody comprising a detectable label, the detection antibody binding to the capture antibody-oAβ complex under conditions sufficient for the capture antibody-oAβ complex to be bound; and detecting and optionally quantifying the detectable label, the amount of detectable label being proportional to the amount of oAβ in the sample, thereby detecting oAβ in the sample.

[0013] In some embodiments, the one or more biological fluids include whole blood, plasma, serum, urine, saliva, exhaled air, exosomes or exosome-like microvesicles, lymph, cerebrospinal fluid, or sputum blood or plasma. Tissues can also be used. Preferably, the sample is whole blood, plasma, or serum. In some embodiments, the sample is diluted. For example, a sample containing plasma is diluted 1:2 to 16 times, such as 1:4 to 1:8 times. A sample containing CSF may be diluted 1:10 to 1:20 times, such as 1:12 to 1:18, such as 1:16 times.

[0014] In some embodiments, the surface is the surface of a plate or bead. In some embodiments, the plate is a multi-well plate (e.g., a 96-well plate) or a test strip. In some embodiments, the bead is selected from magnetic beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, such as SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, or TEFLON® beads.

[0015] In some embodiments, the capture antibody is conjugated to a biotin moiety, the surface is coated with streptavidin, and the antibody is attached to the surface via a biotin-streptavidin bond.

[0016] In some embodiments, the detection antibody binds to Aβ at a different epitope than the capture antibody.

[0017] In some embodiments, the capture antibody comprises an affinity tag and the detection antibody binds to the affinity tag.

[0018] In some embodiments, the detectable label is a radioisotope, a chemiluminescent agent, a fluorescent agent, a metal complex, a bioluminescent agent such as luciferase, or a nucleic acid.

[0019] In some embodiments, the detection and optional quantification of the oAβ concentration in the sample includes using a radioimmunoassay (RIA) when the detection antibody is labeled with a radioisotope, using a chemiluminescent immunoassay (CIA) when the detection antibody is labeled with a chemiluminescent substance, using a fluorescent immunoassay (FIA) when the detection antibody is labeled with a fluorescent substance, using an electrochemiluminescent immunoassay (ECLIA) when the detection antibody is labeled with a metal complex, using a bioluminescent immunoassay (BLIA) when the detection antibody is labeled with a bioluminescent substance such as luciferase, using immunoPCR including amplifying the antibody-labeled nucleic acid by PCR and detecting the nucleic acid, using a turbidimetric immunoassay (TAI) that detects turbidity caused by the formation of an immune complex, using a latex agglutination turbidimetric assay (LA) that includes detecting latex aggregated by the formation of a complex, or using an immunochromatographic assay using a reaction on a cellulose membrane.

[0020] In addition, the present specification provides a kit comprising a surface, optionally a plate or bead; a capture antibody selected from 71A1 or 1G5, optionally linked to the surface; a detection antibody; and a standard comprising a known concentration of oAβ. In some embodiments, the surface is a plate or bead surface. In some embodiments, the plate is a multi-well plate or a test strip.

[0021] In some embodiments, the beads are selected from magnetic beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, such as SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, or TEFLON® beads.

[0022] In some embodiments, the capture antibody is conjugated to a biotin moiety, the surface is coated with streptavidin, and the antibody is attached to the surface via a biotin-streptavidin bond.

[0023] In some embodiments, the detection antibody binds to Aβ at a different epitope than the capture antibody.

[0024] In some embodiments, the capture antibody comprises an affinity tag and the detection antibody binds to the affinity tag.

[0025] In some embodiments, the detectable label is a radioisotope, a chemiluminescent agent, a fluorescent agent, a metal complex, a bioluminescent agent such as luciferase, or a nucleic acid.

[0026] Also provided herein is a method for treating a neurological disorder associated with accumulation of oAβ in a subject, comprising administering to the subject a therapeutically effective amount of 71A1 antibody or an antigen-binding fragment thereof or 1G5 antibody or an antigen-binding fragment thereof. Also provided are 71A1 antibody or an antigen-binding fragment thereof or 1G5 antibody or an antigen-binding fragment thereof, and compositions comprising 71A1 antibody or an antigen-binding fragment thereof or 1G5 antibody or an antigen-binding fragment thereof, for example, for use in a method for treating a neurological disorder associated with accumulation of oAβ in a subject. In some embodiments, the disorder is cerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD). In some embodiments, the antibody is administered to the brain of the subject. In some embodiments, the antibody is a monoclonal antibody, a humanized antibody, or a chimeric antibody.

[0027] Provided herein is a quantitative method for assaying a target analyte present in one or more biological fluids. Such a method comprises the steps of: (i) obtaining a fluid sample from a human patient; (ii) capturing the target analyte present in the fluid with a first agent or probe (capture probe) that recognizes and isolates the target analyte from the fluid; and (iii) quantifying the concentration of the analyte in the biological fluid using a second agent or probe (detector probe) that recognizes a different epitope of the target analyte and is conjugated with a detector signal, wherein the target analyte is quantified by the detector signal. In some embodiments, the target analyte is a soluble neurotoxic multimeric antigen (oAβ) derived from amyloid beta protein (Aβ). In some embodiments, the oAβ is composed of Aβ peptide isoforms. In some embodiments, the Aβ peptide isoforms are Aβ peptides that are naturally secreted into biological fluids. In some embodiments, the naturally secreted Aβ peptides include As37, As38, As39, Aβ40, Aβ42, As43, and any N-terminal variants thereof. In some embodiments, the capture and detector probes include monoclonal antibodies, derivatives of monoclonal antibodies, modifications of monoclonal antibodies, or polyclonal antibodies. In some embodiments, the capture monoclonal antibodies are oAβ selective and recognize and bind to soluble multimers composed of any of the As peptides described herein. In some embodiments, the capture monoclonal antibody is 71A1 or 1G5.9. In some embodiments, the capture probe or monoclonal antibody is conjugated to a complementary binding molecule. In some embodiments, the complementary binding molecule includes, but is not limited to, biotin. In some embodiments, the capture probe is bound to a substrate or surface. In some embodiments, the substrate or surface is coated with streptavidin.In some embodiments, the substrate or surface of the capture probe includes beads, particles, microspheres, nanotubes, polymers, plates, disks, or dipsticks. In some embodiments, the type of beads can be characterized as magnetic beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, such as SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, or TEFLON beads. In some embodiments, the detector probe or monoclonal antibody recognizes an N-terminal epitope of Aβ. In some embodiments, the detector probe or monoclonal antibody includes, but is not limited to, 3D6. In some embodiments, the detector probe or monoclonal antibody is conjugated to, but is not limited to, an enzyme label, a dye, a fluorescent label, or a metal label. In some embodiments, the fluorescent label is a bright near-infrared fluorescent dye that ideally exhibits suitable excitation for 594 nm or 633 nm laser lines. In some embodiments, the fluorescent label is measured using a confocal microscope lens and a photodetector.

[0028] Also provided herein is a non-invasive method for screening a human patient for the presence and course of a neurological disease or condition characterized by the deposition and / or production of soluble neurotoxic Aβ multimers, comprising: (i) quantifying the amount of soluble neurotoxic Aβ multimer antigen in a bodily fluid from said human patient using the methods described herein; (ii) comparing the amount of soluble neurotoxic Aβ multimers in said human patient sample with the amount of soluble neurotoxic Aβ multimers in a fluid sample isolated from a normal human reference population, and (iii) a change in the amount of soluble neurotoxic Aβ multimers present in said human patient fluid sample compared to the amount of soluble multimers present in the fluid sample of said normal human reference population is a biomarker or diagnostic for the presence and / or course of said neurological disease or condition. In some embodiments, the non-invasive method screens for a biomarker for the effectiveness of a therapeutic intervention for said neurological disease or condition.

[0029] Further provided herein is a kit for quantifying the amount of soluble neurotoxic Aβ multimeric antigen in a sample obtained from a human patient, the kit comprising (i) capture and detector reagents as described herein, and (ii) instructions for using said reagents to quantify soluble neurotoxic Aβ multimeric antigen in said human patient sample.

[0030] Additionally, provided herein is a method for preventing, slowing, or treating a disease characterized in part or in whole by pathological extracellular deposition of amyloid in a patient, comprising administering to said patient one or more therapeutically effective doses of an agent that inhibits neurotoxic multimers of amyloid beta protein (oAβ). In some embodiments, the agent is a monoclonal antibody. In some embodiments, the monoclonal antibody is 71A1 or 1G5. In some embodiments, the agent is administered by the following routes, including but not limited to: oral, sublingual, parenteral, intraperitoneal, intramuscular, intravenous, topical, intraocular, intranasal, intracerebral, intraventricular, and intracisternal. In some embodiments, the disease is a neurodegenerative disease, such as Alzheimer's disease.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in the present invention are described herein. Other suitable methods and materials known in the art can also be used. Materials, methods, and examples are for illustration only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of discrepancy, the present specification, including definitions, will control.

[0032] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram of an exemplary peptide immunogen, also referred to herein as NETL-1. In this example, the peptide is shown conjugated to a maleimide carrier. [Diagram 2]Standard enzyme-linked immunosorbent assay (ELISA) using purified monoclonal antibodies assayed for binding to Aβ40, NETL1, or NETL2 peptides conjugated to maleimide-activated (SH-reactive) microtiter plates. [Figure 3-1] 3A-3D: 1G5 and 71A1 pull down Aβ from human brain soaked extracts. (A) Schematic for preparing human brain soaked extracts. [Figure 3-2] 1G5 and 71A1 pull down Aβ from human brain soaked extracts. (B) Immunoblot of protein G immunoprecipitation from human brain soaked extracts with different antibodies. Detection was with two antibodies against the mid-region and N-terminus of Aβ. 7.5 μL of extract was used in the input lane. IPs were from 800 μL of extract. [Figure 3-3] 1G5 and 71A1 pull down Aβ from human brain soaked extracts. (C) Aβx-40 and x-42 measured by immunoprecipitation with the indicated antibodies from human brain soaked extracts or by ELISA of post-immunoprecipitation supernatants (all denatured with 8 M GnCl). n=3, mean ± SD. [Diagram 3-4] 1G5 and 71A1 pull down Aβ from human brain soaked extracts. [Figure 4-1] 4A-4D: 1G5 and 71A1 recognize Aβ in situ in human brain. (A) Immunohistochemical characterization using 1C22, 1G5, and 71A1 on human brain cryosections. Scale bar = 200 μm. [Figure 4-2] 1G5 and 71A1 recognize Aβ in situ in human brain. (B) Immunohistochemical characterization of human brain cryosections using 1C22 or 71A1, double-labeled with Aβ monomer antibody D54D2. Bars = 200 μm in the two left panels, 100 μm in the right panel. [Figure 4-3]1G5 and 71A1 recognize Aβ in situ in human brain. (C) LTP induction following treatment with aCSF (n=6), human brain soaked extract (n=5), 2.12ug / mL 71A1 (n=4), or human brain soaked extract premixed with 2.12ug / mL 71A1 (n=4). Mean ± SD. (D) LTP induction following treatment with aCSF (n=6) or 71A1 affinity purified oAβ (n=4). Mean ± SD. [Figure 4-4] Figures 4E-4F: Statistical tests of electrophysiological recordings on hippocampal slices. (E) Multiple t-test analysis of human brain immersion extract treatments versus the same extract treatment premixed with 2.12ug / mL 71A1 (Figure 4C): q-value distribution over 60 minutes of recording. [Figure 4-5] Statistical tests of electrophysiological recordings on hippocampal slices. (F) Multiple t-test analysis between aCSF treatment vs. 71A1 purified oAβ treatment (Figure 4D): q-value distribution over 60 min of recording. [Figure 5-1] Figures 5A-5C: 1G5 and 71A1 recognize Aβ from human CSF. (A) Aβx-40 and x-42 measured by ELISA on immunoprecipitates or post-immunoprecipitation supernatants from three human CSFs with different antibodies (all denatured with 8 M GnCl). Technical replicates = 3, mean ± SD. [Figure 5-2] (Continued from Figure 5A) 1G5 and 71A1 recognize Aβ from human CSF. [Figure 5-3] 1G5 and 71A1 recognize Aβ from human CSF. (B) Aβx-40 and x-42 measured by ELISA of 71A1 immunoprecipitates or post-immunoprecipitation supernatants (all denatured with 8 M GnCl) from 19 individual human CSFs. Technical replicates = 3, mean ± SD. [Figure 5-4] 1G5 and 71A1 recognize Aβ from human CSF. (C) Correlation between Aβx-40 and x-42 measured by ELISA on 71A1 immunoprecipitates (all denatured with 8 M GnCl) and ADmark Aβ1-42 measurements from the same CSF. Pearson correlation was used. [Figure 6-1]Figures 6A-6I: Development of 71A1 / 3D6 immunoassay specific for oAβ. (A) Schematic of the SMCxPRO bead-based immunoassay. [Figure 6-2] Development of the 71A1 / 3D6 immunoassay specific for oAβ. (B-C) 1G5 / 3D6 and 71A1 / 3D6 assay performance with ADDLs as calibrators. (D) Mean CV and recovery of ADDL signal using the 1G5 / 3D6 and 71A1 / 3D6 assays. [Figure 6-3] Development of 71A1 / 3D6 immunoassays specific for Aβ. (E) 1C22 / 3D6 and 71A1 / 3D6 assay performance against ADDLs as calibrators. (F) CBB staining of PAGE analysis of Aβ1-40 S26 dimers without (-) or with (+) reduction with DTT. [Figure 6-4] Development of 71A1 / 3D6 immunoassays specific for Aβ. (GH) 1C22 / 3D6 and 71A1 / 3D6 assay performance on Aβ1-40 S26 dimer without (native) or with reduction with DTT. (I) 71A1 / 3D6 assay performance on human brain soaked extracts: Left panel: raw 71A1 / 3D6 signal from serially diluted brain extracts calibrated with ADDLs; Middle panel: calculated values ​​adjusted by dilution factor; Right panel: recovery of each dilution normalized to 1:1K; n=3, mean ± SD. [Figure 6-5] (Continued from Figure 6I ) Development of the 71A1 / 3D6 immunoassay specific for oAβ. [Figure 7-1] 7A-7D: 71A1 / 3D6 immunoassay recognizes high molecular weight oAβ from human brain. (A-B) Chromatograms (UV 280 nm absorbance) of molecular weight calibrators and human brain soluble extracts fractionated on a Superdex200 increase SEC column. [Figure 7-2] The 71A1 / 3D6 immunoassay recognizes high molecular weight oAβ from human brain. (C) Native oAβ measured by 71A1 / 3D6 immunoassay from SEC fractions. n=3, mean ± SD. [Figure 7-3]The 71A1 / 3D6 immunoassay recognizes high molecular weight oAβ from human brain. (D) Native (red bars) or denatured (blue bars) monomeric Aβx-42 measured by MSD ELISA from SEC fractions. n=2, mean ± SD. [Figure 8-1] 8A-8F: 71A1 / 3D6 immunoreactive oAβ in CSF correlates with t-tau and p-tau levels. (A) 71A1 / 3D6 assay performance on human CSF: Left panel: raw 71A1 / 3D6 signal (relative to ADDL calibrators) from serially diluted CSF; Middle panel: calculated values ​​adjusted by dilution factor; Right panel: percent recovery of each dilution normalized to 1:2; n=3, mean ± SD. [Figure 8-2] 71A1 / 3D6 immunoreactive oAβ in CSF correlates with t-tau and p-tau levels. (B-C) Correlation between 71A1 / 3D6 reactive oAβ levels in CSF and ADmark measurements of Aβ1-42, t-tau, and p-tau. n=29. Pearson correlation was used. [Figure 8-3] 71A1 / 3D6 immunoreactive oAβ in CSF correlates with t-tau and p-tau levels. (D) Correlation between 71A1 / 3D6 reactive oAβ levels in CSF and ADmark measurements of Aβ1-42, t-tau, and p-tau. n=29. Pearson correlation was used. [Figure 8-4] 71A1 / 3D6 immunoreactive oAβ in CSF correlates with t-tau and p-tau levels. (E) Chromatogram (UV 280 nm absorbance) of human CSF fractionated on a Superdex 200 Increase SEC column. [Figure 8-5] 71A1 / 3D6 immunoreactive oAβ in CSF correlates with t-tau and p-tau levels. (F) Native oAβ measured by 71A1 / 3D6 immunoassay in SEC fractions. n=3, mean±SD. [Figure 9-1]9A-9G: 71A1 / 3D6 immunoassay accurately quantifies oAβ from human plasma. (A) 71A1 / 3D6 assay performance on human plasma: Left panel: raw 71A1 / 3D6 signal from serially diluted plasma against ADDL calibrators; Middle panel: calculated values ​​adjusted by dilution factor; Right panel: recovery of each dilution normalized to 1:4; n=3, mean ± SD. [Figure 9-2] The 71A1 / 3D6 immunoassay accurately quantifies oAβ from human plasma. (B) Spike-in recovery studies by spiking human brain homogenate (Brain H), human brain macerated extract (Brain S), or human CSF into individual plasma samples diluted 8-fold. n=3, mean ± SD. [Figure 9-3] The 71A1 / 3D6 immunoassay accurately quantifies oAβ from human plasma. (C) 71A1 / 3D6 assay of oAβ from 8-fold dilutions of individual plasma immunodepleted with various antibodies compared to input plasma. n=4. [Figure 9-4] The 71A1 / 3D6 immunoassay accurately quantifies oAβ from human plasma. (D) 71A1 / 3D6 assay of oAβ from human brain immersion extracts, CSF, and plasma denatured (or not) with 8 M GnCl. n=3, mean ± SD. [Figure 9-5] The 71A1 / 3D6 immunoassay accurately quantifies oAβ from human plasma. (E) 71A1 / 3D6 assay of oAβ from 73 human plasma samples (Mayo Elderly Cohort), n=4. [Figure 9-6] The 71A1 / 3D6 immunoassay accurately quantifies oAβ from human plasma. (F) Raw 71A1 / 3D6 signal (relative to ADDL calibrators) from serially diluted plasma; n=3, mean ± S. [Figure 9-7] The 71A1 / 3D6 immunoassay accurately quantifies oAβ from human plasma. (G) Recovery of each dilution normalized to the mean of all dilutions; n=3, mean ± SD. [Figure 10-1]FIG. 10A. Quantification of oAβ in APP NLGF / NLGF mouse brain soaked extracts shows age-dependent accumulation of 71A1-positive oAβ in mouse brain. [Figure 10-2] FIG. 10B. Measurement of long-term potentiation (LTP) in the hippocampus of 6-month-old APP NLGF / NLGF mice and age-matched wild-type mice shows impaired LTP function in 6-month-old APP NLGF / NLGF mice. [Figure 10-3] Figure 10C. The immersion extract from 6-month-old APP NLGF / NLGF mice was used to treat wild-type mouse hippocampus, followed by LTP measurement. The 6-month-old APP NLGF / NLGF mouse brain extract could impair LTP in treated wild-type mouse hippocampus. Mixing 71A1 in the treatment also rescued the dysfunction caused by the 6-month-old APP NLGF / NLGF mouse brain extract. [Figure 11-1] Figure 11A. Y-maze behavior of mice treated with 71A1 or control antibody, as indicated by both spontaneous alteration and arm entries. 71A1 treatment significantly improved the performance of male mice, but not females, compared to control antibody treatment. [Figure 11-2] (Continued from FIG. 11A). Y-maze behavior of mice treated with 71A1 or control antibody, as indicated by both spontaneous alteration and arm entries. 71A1 treatment significantly improved the performance of male mice, but not females, compared to control antibody treatment. [Figure 11-3] FIG. 11B. 71A1 treatment reduced oAβ levels in the brain of male mice (trend without statistical significance). [Figure 12-1] FIG. 12A. Schematic representation of LTP measurements in wild-type mouse hippocampus treated with brain extracts from antibody test APP NLGF / NLGF mice. [Figure 12-2]Figure 12B. Wild-type mouse hippocampi were treated with brain extracts from 71A1-treated (n=3) and control antibody-treated (n=2) male mice to show that 71A1 treatment reduced synaptic toxicity in APP NLGF male mouse brains, as reflected by its failure to inhibit LTP in wild-type mouse hippocampi. [Figure 13] The sequences of 12 synthetic peptidomimetics that bind to Mab71A1, isolated using New England BioLab's Ph.D.-12™ Phage Display Peptide Library Kit, are shown in order as SEQ ID NOs: 5 to 17. [Figure 14-1] 14A-14B: ELISA using purified monoclonal antibody 71A1 assayed for binding to peptidomimetics (A) 1-7 and (B) 8-13 conjugated to maleimide-activated microtiter plates as shown in FIG. 13. [Figure 14-2] 14A-14B: ELISA using purified monoclonal antibody 71A1 assayed for binding to peptidomimetics (A) 1-7 and (B) 8-13 conjugated to maleimide-activated microtiter plates as shown in FIG. 13. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Biochemical and morphological studies suggest that clinical impairment in AD is accompanied by early synaptic dysfunction (Tu et al., 2014;Anderton et al., 1998;Cummings et al., 1998), followed by more severe neuronal changes including increased synaptic loss, widespread neuritic dystrophy, neurofibrillary tangles, and frank neuronal death (Serrano-Pozo et al., 2011;Tonnies & Trushina, 2017;Terry, 1963;Gomez-Isla et al., 1996;Sze et al., 1997;Anderton et al., 1998). Mechanisms underlying the initiation of this progressive pathophysiology are thought to involve age-related dysregulation and accumulation of amyloid-β protein (Aβ) and subsequent hyperphosphorylation and aggregation of the microtubule-associated protein tau, both of which can be observed at autopsy in fibril-rich senile plaques and neurofibrillary tangles, respectively (Braak & Braak, 1996;Perl, 2010;Esiri et al., 1997). The observation of these end-stage lesions in postmortem brain tissue, and the appearance of amyloid plaques before neurofibrillary tangles, have led to the presumption that fibril accumulation itself underlies the progression of AD (Serrano-Pozo et al., 2011). This impression has been partially supported by studies in primary neuronal cultures, where progressive neurodegeneration can be induced by highly aggregated fibrillar Aβ but not by comparable concentrations of Aβ monomers (Mattson et al., 1993b;Pike et al., 1993;Lorenzo et al., 1994). Moreover, several studies have shown that Aβ dysregulation likely precedes and induces tau hyperphosphorylation, ultimately leading to the clinical symptoms of the disease (Zheng et al., 2002;Wu et al., 2018;Bloom 2014;Viola and Klein, 2015).

[0035] Alzheimer's disease (AD) is characterized by the formation of fibril-rich plaques composed of amyloid b protein (Aβ) that were thought to disrupt normal communication between brain cells and ultimately lead to neuronal death. Although the accumulation of amyloid plaques was once thought to be the sole cause of the neuronal toxicity found in AD, small soluble aggregates of beta-amyloid may be more toxic (Hong et al., 2018; Cline et al., 2018).

[0036] Aβ is a peptide fragment of a larger protein called amyloid precursor protein or APP. A portion of APP is expressed on the surface of neurons, which is then cleaved to produce a 40- or 42-amino acid fragment called Aβ. Multimeric Aβ assemblies, as well as larger intermediates called protofibrils (PFs), form during fibrilogenesis. In vitro studies have shown that soluble multimeric species of Aβ and protofibrils can alter neurophysiology and exhibit neurotoxicity, strongly suggesting that such assemblies may play a role in the pathophysiology of AD (Cleary et al, 2005; Hon et al, 2018; Shankar et al, 2018; O'Malley et al, 2016). The development of antibodies that can specifically recognize the multimeric form of Aβ will be invaluable in understanding the central role that multimeric forms play in the initiation of brain dysfunction and injury. In particular, these antibodies will be useful in identifying when these assemblies occur and how they may correlate with memory impairment and cognitive decline in transgenic mice and AD patients. In addition, if protofibrils or unique small aggregates of Aβ form in humans, these novel antibodies may be valuable in identifying such assemblies in blood and / or tissues and aid in the diagnosis of early stages of AD.

[0037] Aβ is believed to begin accumulating in vivo as low molecular weight species (LMW Aβ) consisting essentially of monomers that are constitutively secreted from brain cells (Haass et al., 1992;Shoji et al., 1992). Under certain circumstances, the LMW species may progress to multimers and ultimately to mature amyloid fibrils 7-10 nm wide, as observed in mature plaques. Aβ multimers (dimers, trimers, tetramers, and possibly larger assemblies) have been identified in the conditioned medium of certain cell lines overexpressing APP, which constitutively secrete Aβ (Podlisny et al., 1995;Xia et al., 1997;Podlisny et al., 1998) and in the cerebrospinal fluid of AD patients (Pitschke et al., 1998;Walsh et al., 2000). Recent data from several laboratories also suggest that multimeric species formed during Aβ fibril formation may contribute to neuronal injury (Hong et al., 2018; Li et al., 2018). For example, Aβ complexes that sediment more slowly than amyloid fibrils were shown to have neurotoxic effects on cultured PC12 cells as quantified by MTT conversion assay (Oda et al., 1995). In addition, β-1-antichymotrypsin can block fibril formation but does not reduce Aβ-induced neurotoxicity (Aksenova et al., 1996). Soluble Aβ multimeric species isolated from AD cerebral cortex that kill neurons in mixed brain cultures have also been reported (Giulian et al., 1996; Roher et al., 1996). Furthermore, recently reported soluble forms of synthetic Aβ, termed Aβ-derived diffusible ligands (ADDLs), are neurotoxic in vitro (Lambert et al., 1998).In support of this prefibrillar concept, transgenic mice overexpressing human APP exhibit behavioral and / or electrophysiological changes before any substantial Aβ deposition or neuropathology is observed (Holcomb et al., 1998;Chapman et al., 1999;Hsia et al., 1999).These data clearly suggest that the prefibrillar form of Aβ may cause synaptic dysfunction and thus contribute to the early memory loss and mild cognitive changes in the early stages of AD. Interestingly, a recent human study found that the best correlation between mental status and synaptic loss in AD patients was the amount of soluble Aβ. However, the biophysical properties of the soluble Aβ species were not determined (Lue et al., 1999).

[0038] As mentioned above, fibril formation is thought to proceed via the transition of LMW Aβ to metastable intermediate species that progress to the formation of fibrils (Harper et al., 1997a;Teplow, 1998). Two laboratories have identified such intermediates in the formation of synthetic Aβ fibrils, which they have termed protofibrils (PF) (Harper et al., 1997a;Harper et al., 1997b;Walsh et al., 1997). Laboratories have investigated the electrophysiology and neurotoxicity of both low molecular weight Aβ (LMW) (i.e., monomers / dimers) and PF, as well as their relationship to fibrillar Aβ (Hartley et al., 1999). Both of these earlier species were found to reproducibly induce toxicity over several days in cultured primary cortical neurons. This toxicity was not associated with the detectable appearance of amyloid fibrils, as measured by Congo Red binding and immunoelectron microscopy (Hartley et al., 1999). Consistent with possessing intrinsic biological activity, PF robustly and reproducibly increased excitatory postsynaptic currents (EPSCs), excitatory postsynaptic potentials (EPSPs), action potentials (APs), and membrane depolarization (MD) (Hartley et al., 1999). Importantly, PF exerted such effects at doses as low as 50-100 nM, whereas monomeric Aβ, prepared and applied simultaneously at the same concentrations, was found to elicit no electrophysiological response. These effects of PF were fully reversible within 65 min after application (see Ye et al., 2000).

[0039] Elucidating the biological activity of PF should help determine the role of early Aβ intermediates in the mechanism of neuronal dysfunction in AD and the associated therapeutic implications. Interestingly, aggregation of other proteins may play an important role in polyglutamine expansion disorders such as Parkinson's disease, prion diseases, Huntington's disease, and frontotemporal dementia (Kim et al., 1999;Lansbury et al., 2000). Thus, understanding the details of early Aβ aggregation and its associated neuronal dysfunction can serve as a prototype for understanding other brain diseases involving protein aggregation. Therefore, reagents such as protofibril-specific antibodies, which are important for identifying pathological species, will be invaluable in determining their role in such neurodegenerative diseases.

[0040] Studies of amyloid protofibrils and amyloid fibrils show a characteristic β-sheet structure in which the polypeptide chains run nearly perpendicular to the long axis of the fibril, with interchain hydrogen bonds parallel to the axis. It is the amyloid fibrils that form the “senile plaques” in Alzheimer’s disease. How the fibrils form and the exact nature of the early events that lead to fibril formation are less well understood. β-sheet intermediates of Aβ peptides, often called protofibrils, form during the process of fibril formation. If such structures could be immunologically detected early in disease development, it could be useful in determining their role in AD pathogenesis. Furthermore, such early assemblies, if at least partially involved in the development of AD, could potentially be used as diagnostic markers and contribute to the development of therapeutic agents that target such structures. Antibodies that detect protofibrils could be invaluable in this endeavor and would be useful in monitoring therapy efficacy. Several studies suggest that passive immunization of AD animal models with anti-Aβ antibodies can reduce Aβ burden and reverse behavioral deficits (Morgan, 2011). This may indicate that passive immunization with anti-Aβ antibodies against early pathological species may be of great value for early intervention in AD.

[0041] Peptide Immunogens Described herein are synthetic peptide immunogens, e.g., peptides optionally 6-50 amino acids in length, e.g., at least 6, 7, 8, 9, 10, 11, or 12 amino acids, and up to 15, 20, 25, 30, 35, 40, 45, or 5 amino acids, as well as structures and sequences of regions within Aβ peptides that can be used to facilitate the development of antibodies that bind to Aβ aggregates but not to normally occurring monomeric Aβ amino acid peptides. In some embodiments, the synthetic peptide immunogen comprises the Aβ peptide sequence GYEVHHQKLV (SEQ ID NO: 1).

[0042] In some embodiments, the synthetic peptide immunogen comprises the following peptide sequence: CGKCGYEVHHQKLVPNVLKQHHVEYGCK (SEQ ID NO: 2)

[0043] Peptide immunogens are useful, for example, for the generation of isolated and purified antibodies, including, but not limited to, the IgG subclass 1 monoclonal antibodies 71A1 and 1G5.

[0044] Additionally, peptide immunogens are provided herein that contain peptidomimetic sequences that bind to the 71A1 variable region. Such peptidomimetics are linear peptide sequences that mimic part of the three-dimensional structure of the Abeta multimer specific to mab 71A1 or 1g5, and thus mimic the natural antigen structure with a linear sequence that also binds to these antibodies (see FIG. 13).

[0045] Such peptidomimetics were affinity selected using monoclonal antibody 71A1 and a commercially available phage display library kit (see Example 6). Thus, peptides including one of the sequences shown in Figure 13, e.g., SEQ ID NOs: 5-17, are also provided.

[0046] [Table 1]

[0047] In some embodiments, the peptide immunogen comprises the sequence YEXHH (SEQ ID NO:41), where X is a hydrophobic amino acid (V, I, or L). In some embodiments, the peptide immunogen comprises the sequence GYEVHHQKLV (SEQ ID NO:1), or a sequence that is at least 80, 85, 90, 95, or 99% identical thereto, e.g., containing mutations at 1, 2, or 3 amino acids.

[0048] The peptides and peptide immunogens of the present invention can be produced by chemical synthesis methods well known to those skilled in the art. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman & Co., New York, NY, 1992, p. 77. Thus, peptides can be synthesized, for example, on an Applied Biosystems peptide synthesizer model 430A or 431, using automated Merrifield techniques of solid-phase synthesis with alpha-NH2 protected by either t-Boc or F-moc chemistry using side-chain protected amino acids. Preparation of peptide constructs, including combinatorial library peptides against Th epitopes, can be accomplished by providing a mixture of alternative amino acids for coupling at a given variable position. After the desired peptide immunogen is fully assembled, the resin is treated according to standard procedures to cleave the peptide from the resin and deblock the functional groups of the amino acid side chains. The free peptides are purified by HPLC and biochemically characterized, for example, by amino acid analysis or sequencing. Methods for purifying and characterizing peptides are well known to those skilled in the art.

[0049] The peptides and peptide immunogens can be used, for example, to generate antibodies that bind to Aβ, e.g., Aβ aggregates (oAβ), to elicit an immune response, e.g., in an animal. Methods for producing antibodies are known in the art and described herein. Also provided herein are methods for using the peptides and peptide immunogens described herein, and compositions that include the peptides and peptide immunogens described herein. In some embodiments, the composition includes an adjuvant. In some embodiments, the adjuvant is covalently linked to the peptide. In some embodiments, the peptide is covalently linked to a carrier that includes Tepi-2 (SEQ ID NO: 4), or keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or ovalbumin (OVA).

[0050] Antibodies 71A1 and 1G5 As described above, the present inventors have succeeded in obtaining an antibody that specifically binds to Aβ multimers but does not bind to Aβ monomers. That is, the present disclosure provides an antibody that binds to Aβ multimers but does not bind to Aβ monomers.

[0051] Antibodies useful in the methods and compositions of the invention include 71A1 and 1G5, and variants and derivatives thereof. Antibody 71A1 Heavy Chain: DNA sequence

[0052] [ka] Predicted protein sequence (complementarity determining regions (CDRs) are in bold)

[0053] [ka] Light chain: DNA sequence

[0054] [ka] Predicted protein sequence (complementarity determining regions (CDRs) are in bold)

[0055] [ka] Antibody 1G5 Heavy Chain: DNA sequence

[0056] [ka] Predicted protein sequence (complementarity determining regions (CDRs) are in bold)

[0057] [ka] Light chain: DNA sequence

[0058] [ka] Predicted protein sequence (complementarity determining regions (CDRs) are in bold)

[0059] [ka]

[0060] antibody Antibodies of the present disclosure can include any type of antibody, such as unmodified (native) monoclonal antibodies, non-human animal antibodies, humanized antibodies, chimeric antibodies, human antibodies, minibodies, bispecific antibodies (e.g., those that bind to oAβ and receptors involved in RMT such as the transferrin receptor (TfR), insulin receptor (IR), or low density lipoprotein receptor-related protein-1 (LRP-1)), amino acid sequence-modified antibodies, modified antibodies conjugated to other molecules (e.g., polymers such as polyethylene glycol), and glycosylated antibodies, as well as antigen-binding fragments thereof.

[0061] The antibodies used in the methods and compositions of the present invention are preferably isolated or purified. The terms "isolated" and "purified" as used with respect to the substances described herein indicate that the substances are substantially free of at least one other substance that may be contained in the natural source. Thus, "isolated antibody" and "purified antibody" refer to an antibody that is substantially free of cellular material such as carbohydrates, lipids, or other contaminating proteins from the cell or tissue source from which the antibody (protein) is derived. If the antibody is chemically synthesized, the term refers to an antibody that is substantially free of chemical precursor materials or other chemicals. In a preferred embodiment, the antibodies provided herein are isolated or purified.

[0062] "Monoclonal antibody", as used herein, is intended to refer to one of a preparation of antibody molecules that includes antibodies sharing a common heavy chain amino acid sequence and a common light chain amino acid sequence, as opposed to antibodies of a "polyclonal" antibody preparation that includes a mixture of different antibodies. Monoclonal antibodies can be produced by several new technologies such as phage, bacterial, yeast, or ribosome display, as well as classical methods exemplified by hybridoma-derived antibodies (e.g., antibodies secreted by hybridomas prepared by hybridoma technology such as standard Kohler and Milstein hybridoma technology ((1975) Nature 256:495-497)). Thus, non-hybridoma-derived antibodies are still referred to as monoclonal antibodies, even if they may have been obtained by non-classical methodologies. In addition to the specificity mentioned above, monoclonal antibodies have the advantage that they can be synthesized from hybridoma cultures free of other immunoglobulin contamination. Thus, "monoclonal" refers to the quality of an antibody that can be obtained from a substantially homogeneous antibody population. The term does not imply a requirement of any particular method for producing the antibody.

[0063] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a globulomer is substantially free of antibodies that specifically bind to other antigens other than the globulomer). However, an isolated antibody that specifically binds to a globulomer may exhibit cross-reactivity to other antigens. Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0064] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be in a bispecific, bispecific, or multispecific format that specifically binds to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment comprising a single variable domain (Ward et al., (1969) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but may be joined by a synthetic linker that allows them to be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but because a linker that is too short to allow pairing between the two domains on the same chain is used, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. j., et al. (1994) Structure 2:1121-1123). Such antibody-binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

[0065] Furthermore, an antibody or an antigen-binding fragment thereof may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques as described herein.

[0066] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, e.g., an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0067] The term "CDR-grafted antibody" refers to an antibody that comprises heavy and light chain variable region sequences from one species, but in which one or more sequences of the VH and / or VL CDR regions have been replaced with CDR sequences from another species, e.g., an antibody having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0068] The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In particular, the term "humanized antibody" refers to an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Also preferably, the humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. In some embodiments, the humanized antibody comprises both a light chain as well as at least the variable domains of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In other embodiments, the humanized antibody comprises only a humanized heavy chain. In certain embodiments, the humanized antibody comprises only a humanized variable domain of the light chain and / or a humanized heavy chain.

[0069] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0070] The framework and CDR regions of a humanized antibody do not have to correspond exactly to the parental sequences, for example, the donor antibody CDR or consensus framework may be mutated by substitution, insertion, and / or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in a preferred embodiment, such mutations will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to the residues of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region of a consensus immunoglobulin sequence. Furthermore, as used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) which occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position of the consensus sequence is occupied by the amino acid which occurs most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.

[0071] The term "recombinant human antibody," as used herein, refers to an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), an antibody isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL, (1999) Immunology Today 21:371-378), or an antibody isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL, (1999) Immunology Today 21:371-378). (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences, are intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.(See also Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No 91-3242, 1991.) However, the human antibodies provided herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). (See also Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990).

[0072] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, e.g., an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0073] The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species, but in which one or more sequences of the VH and / or VL CDR regions have been replaced with CDR sequences from another species, e.g., an antibody having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0074] The recombinant human antibodies provided herein have variable regions derived from human germline immunoglobulin sequences and may also include constant regions. (See Kabat et al. (1991) supra.) However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo. However, in certain embodiments, such recombinant antibodies are the result of selective mutagenesis or back mutation, or both.

[0075] The term "backmutation" refers to a process in which some or all of the somatically mutated amino acids of a human antibody are replaced with the corresponding germline residues of a homologous germline antibody sequence. The heavy and light chain sequences of the human antibodies described herein are aligned separately with the germline sequences in the VBASE database to identify the sequences with the highest homology. VBASE is a comprehensive directory of all human germline variable region sequences collected from public sequences including the latest releases of the GenBank and EMBL data libraries. This database was developed at the MRC Protein Engineering Centre (Cambridge, UK) as a repository of sequenced human antibody genes (website: mrc-cpe.cam.ac.uk / vbase-intro.php?menu=901). Differences in the antibodies described herein are reverted to the germline sequence by mutating the defined nucleotide positions that code for such different amino acids. The role of each amino acid thus identified as a candidate for backmutation should be investigated for its direct or indirect role in antigen binding, and any amino acid found after mutation that affects any desired attribute of the human antibody should not be included in the final human antibody. To minimize the number of amino acids that undergo backmutation, an amino acid position that differs from the closest germline sequence but is found to be identical to a corresponding amino acid in a second germline sequence may be left alone as long as the second germline sequence is identical and colinear with the sequence of the human antibody for at least 10, preferably 12 amino acids on either side of the amino acid in question. Backmutations can occur at any stage of antibody optimization.

[0076] A "labeled binding protein" is a protein in which an antibody or antibody portion has been derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein can be derived by functionally linking (by chemical coupling, by genetic fusion, by non-covalent association, or otherwise) an antibody or antibody portion to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0077] As used herein, "glycosylated binding protein" includes proteins in which an antibody or antigen-binding fragment thereof contains one or more carbohydrate residues. In the initial in vivo protein production, further processes known as post-translational modifications may occur. In particular, sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting protein has covalently linked oligosaccharide side chains and is known as a glycosylated protein or glycoprotein. Antibodies are glycoproteins that have one or more carbohydrate residues in the Fc domain as well as in the variable domain. Carbohydrate residues in the Fc domain have a significant effect on the effector functions of the Fc domain and a minimal effect on antigen binding or half-life of the antibody (R, Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variable domain may have an effect on the antigen-binding activity of the antibody. Glycosylation of the variable domain may have a negative effect on antibody binding affinity, possibly due to steric hindrance (Co, MS, et al., Mol. Immunol. (1993) 30:1361-1367), or may result in increased affinity for the antigen (Wallick, SC, et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717 2723). Additionally, glycosylation site mutants can be made in which the O-linked or N-linked glycosylation sites of the binding protein are mutated. One of skill in the art can generate such mutants using standard well-known techniques. Glycosylation site mutants that retain biological activity but have increased or decreased binding activity are also contemplated.

[0078] Additionally, the glycosylation of the antibody or antigen-binding fragment can be modified. For example, an aglycosylated antibody can be produced (i.e., the antibody lacks glycosylation). The glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region glycosylation sites, thereby eliminating glycosylation at that site. Such glycosylation can increase the affinity of the antibody for the antigen. Such techniques are described in further detail in WO 03 / 016466 A2, U.S. Pat. Nos. 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.

[0079] Additionally or alternatively, modified antibodies can be made with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNAc structures. Such alterations in glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with altered glycosylation. (See, e.g., Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP 1,176,195; WO 03 / 035835 and WO 99 / 5434280, each of which is incorporated herein by reference in its entirety.)

[0080] Protein glycosylation depends on the amino acid sequence of the protein of interest and the host cell in which the protein is expressed. Different organisms produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases) and have different available substrates (nucleotide sugars). Due to such factors, protein glycosylation patterns and the composition of glycosyl residues may vary depending on the host system in which a particular protein is expressed. Glycosyl residues useful in the present invention may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine, and sialic acid. Preferably, the glycosylated binding protein comprises glycosyl residues whose glycosylation pattern is human.

[0081] Those skilled in the art know that different protein glycosylation can result in different protein characteristics. For example, the efficacy of a therapeutic protein produced in a microbial host such as yeast and glycosylated using the yeast endogenous pathway can be reduced compared to the efficacy of the same protein expressed in a mammalian cell such as a CHO cell line. Such glycoproteins can also be immunogenic in humans and exhibit reduced in vivo half-life after administration. Specific receptors in humans and other animals can recognize specific glycosyl residues and promote rapid clearance of the protein from the bloodstream. Other adverse effects can include changes in protein folding, solubility, susceptibility to proteases, transport, trafficking, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Thus, the expert may choose a therapeutic protein with a specific composition and pattern of glycosylation, for example, the same or at least similar glycosylation composition and pattern as that produced in human cells or species-specific cells of the intended target animal.

[0082] Expression of glycosylated proteins different from that of its host cell can be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes.A person skilled in the art can use techniques known in the art to generate antibodies or antigen-binding fragments thereof that exhibit human protein glycosylation.For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes, so that the glycosylated proteins (glycoproteins) produced in such yeast strains exhibit protein glycosylation identical to that of animal cells, particularly human cells (US Patent Publication Nos. 20040018590 and 20020137134, and WO 05 / 100584 A2).

[0083] Furthermore, one skilled in the art will appreciate that a library of host cells genetically engineered to express various glycosylation enzymes such that member host cells of the library produce proteins of interest with variant glycosylation patterns can be used to express the proteins of interest. The expert can then select and isolate proteins of interest with specific novel glycosylation patterns. Preferably, the proteins with the specifically selected novel glycosylation patterns exhibit improved or altered biological properties.

[0084] The term "activity" includes activities such as the binding specificity / affinity of an antibody for an antigen.

[0085] For purposes of this disclosure, a "fragment" of a sequence is defined as a contiguous sequence of approximately at least 6, preferably at least about 8, more preferably at least about 10, and even more preferably at least about 15 nucleotides corresponding to a region of a specified nucleotide sequence.

[0086] The term "identity" refers to the relatedness of two sequences, either nucleotide-by-nucleotide or amino acid-by-amino acid, over a particular comparison window or segment. Identity is therefore defined as the degree of similarity, correspondence, or equivalence between the same strand (either sense or antisense) of two DNA segments (or two amino acid sequences). "Percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions where identical bases or amino acids are present in both sequences to obtain the number of matching positions, dividing that number by the total number of positions in the segments being compared, and multiplying the result by 100. Optimal alignment of sequences can be performed by the algorithm of Smith & Waterman, Appl. Math. 2:482 (1981), by the algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the method of Pearson & Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), and computer programs implementing related algorithms (e.g., Clustal Macaw Pileup (Higgins et al., CABIOS. 5L151-153 (1989)), FASTDB (Intelligenetics), BLAST (National Center for Biomedical Information; Altschul et al., Nucleic Acids Research 25:3389-3402 (1997)), PILEUP (Genetics Computer Group, Madison, Wis.), or GAP, BESTFIT, FASTA, and TFASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, Madison, Wis.) (see U.S. Pat. No. 5,912,120).

[0087] For the purposes of this disclosure, "complementarity" is defined as the degree of relatedness between two DNA segments. Complementarity is determined by measuring the ability of the sense strand of one DNA segment to hybridize with the antisense strand of another DNA segment under appropriate conditions to form a double helix. A "complement" is defined as a sequence that pairs with a given sequence according to standard base pairing rules. For example, the sequence AGT of one nucleotide strand is "complementary" to TCA of the other nucleotide strand.

[0088] "Similarity" between two amino acid sequences is defined as the presence of a series of identical and conserved amino acid residues in both sequences. The higher the degree of similarity between two amino acid sequences, the more corresponding, similar, or equivalent the two sequences are. ("Identity between two amino acid sequences is defined as the presence of a series of identical or unaltered amino acid residues in both sequences.") The definitions of "complementarity," "identity," and "similarity" are well known to those skilled in the art.

[0089] "Encoded by" refers to a nucleic acid sequence that encodes a polypeptide sequence, or a portion thereof, that contains an amino acid sequence of at least 3 amino acids, more preferably at least 8 amino acids, and even more preferably at least 15 amino acids from the polypeptide encoded by the nucleic acid sequence.

[0090] Furthermore, a nucleic acid molecule is "hybridizable" to another nucleic acid molecule if a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under appropriate conditions of temperature and ionic strength (see Sambrook et al. "Molecular Cloning: A Laboratory Manual, Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

[0091] The term "hybridization" as used herein generally refers to the hybridization of nucleic acids under suitable conditions of stringency, which is easily apparent to those skilled in the art, depending on the nature of the probe sequence and the target sequence.Hybridization and washing conditions are well known in the art, and the adjustment of conditions according to desired stringency can be easily achieved by changing incubation time, temperature, and / or ionic strength of the solution.See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring harbor Press, Cold Spring harbor, NY, 1989.This document is incorporated herein by reference as mentioned above. (See also Short Protocols in Molecular Biology, ed. Ausubel et al. and Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993), both of which are incorporated herein by reference.) In particular, the choice of conditions is determined by the length of the sequences to be hybridized, particularly the length of the probe sequence, the relative GC content of the nucleic acids, and the amount of mismatch that is to be tolerated. Low stringency conditions are preferred when partial hybridization between strands having lower degrees of complementarity is desired. High stringency conditions are preferred when perfect or near perfect complementarity is desired. For typical high stringency conditions, the hybridization solution contains 6x SSC, 0.01M EDTA, 1x Denhardt's solution, and 0.5% SDS. Hybridization is carried out at about 68 degrees Celsius for about 3-4 hours for cloned DNA fragments and about 12-16 hours for total eukaryotic DNA.For moderate stringency, filter pre-hybridization and hybridization in a solution of 3x sodium chloride, sodium citrate (SSC), 50% formamide (0.1M of this buffer at pH 7.5), and 5x Denhardt's solution can be used. Then, pre-hybridization at 37 degrees Celsius for 4 hours, followed by hybridization at 37 degrees Celsius for a total of 16 hours with an amount of labeled probe equivalent to 3,000,000 cpm, followed by washing with 2x SSC and 0.1% SDS solution, 4 washes at room temperature for 1 minute each, and 4 washes at 60 degrees Celsius for 30 minutes each. After drying, exposure to film. For lower stringency, the temperature of hybridization is reduced to about 12 degrees Celsius below the melting temperature (Tm) of the duplex. Tm is known to be a function of GC content and duplex length as well as the ionic strength of the solution.

[0092] "Hybridization" requires that two nucleic acids contain complementary sequences. However, depending on the stringency of the hybridization, mismatches between bases are possible. As discussed above, the appropriate stringency for hybridization of nucleic acids depends on the length of the nucleic acids and the degree of complementation. Such variables are well known in the art. More specifically, the greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having such sequences. For hybrids longer than 100 nucleotides in length, a formula for calculating Tm has been derived (see Sambrook et al., supra). For hybridization with shorter nucleic acids, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra).

[0093] As used herein, an "isolated nucleic acid fragment or sequence" is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be composed of one or more segments of cDNA, genomic DNA, or synthetic DNA. (A "fragment" of a specified polynucleotide refers to a polynucleotide sequence that comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10 nucleotides, even more preferably at least about 15 nucleotides, and most preferably at least about 25 nucleotides that are identical to or complementary to a region of the specified nucleotide sequence.) Nucleotides (usually found in their 5'-monophosphate form) are referred to by single-letter designations as follows: "A" for adenylic acid or deoxyadenylic acid (RNA or DNA, respectively), "C" for cytidylic acid or deoxycytidylic acid, "G" for guanylic acid or deoxyguanylic acid, "U" for uridylic acid, "T" for deoxythymidylic acid, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.

[0094] The terms "functionally equivalent fragment or subfragment" and "functionally equivalent fragment or subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment that retains the ability to modify gene expression or produce a particular phenotype, regardless of whether the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used to design a chimeric construct to produce a desired phenotype in a transformed plant. The nucleic acid fragment or subfragment, whether it encodes an active enzyme or not, can be linked in the appropriate orientation to a plant promoter sequence to design a chimeric construct for use in co-suppression or antisense.

[0095] The terms "homology", "homologous", "substantially similar" and "substantially corresponding" are used interchangeably herein. These terms refer to nucleic acid fragments in which one or more nucleotide base changes do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the present invention, such as the deletion or insertion of one or more nucleotides, that do not substantially change the functional properties of the resulting nucleic acid fragment compared to the initial unmodified fragment. Thus, as will be appreciated by those skilled in the art, it is understood that the present invention encompasses more than the specific exemplary sequences.

[0096] "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0097] A "native gene" refers to a gene found in nature with its own regulatory sequences. In contrast, a "chimeric construct" refers to a combination of nucleic acid fragments that are not normally found together in nature. Thus, a chimeric construct may contain regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but arranged in a manner different from that normally found in nature. (The term "isolated" means that the sequence has been removed from its natural environment.)

[0098] A "foreign" gene refers to a gene not normally found in the host organism, but which has been introduced into the host organism by gene transfer. Foreign genes can include native genes inserted into a non-native organism or a chimeric construct. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

[0099] "Coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0100] A "promoter" or "regulatory gene sequence" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. This sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Thus, an "enhancer" is a DNA sequence capable of stimulating the activity of a promoter or regulatory gene sequence, and may be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. Promoter sequences may also be located within the transcribed portion of a gene and / or downstream of the transcribed sequence. Promoters may be derived in their entirety from a native gene or may be composed of different elements derived from different promoters found in nature, or may even contain synthetic DNA segments. Those skilled in the art will understand that different promoters can direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. Promoters that cause expression of a gene most of the time in most host cell types are commonly referred to as "constitutive promoters". New promoters of various types useful for plant cells are constantly being discovered. Numerous examples can be found in Okamuro and Goldberg, Biochemistry of Plants 15:1-82 (1989). Furthermore, it is recognized that in most cases the exact boundaries of regulatory sequences have not been completely defined, such that DNA fragments with some mutations may have identical promoter activity.

[0101] An "intron" is an intervening sequence within a gene that does not code for a portion of the protein sequence. Thus, such sequences are transcribed into RNA but then excised and not translated. The term is also used for excised RNA sequences. An "exon" is a portion of a gene sequence that is transcribed and found in the mature messenger RNA derived from that gene, but is not necessarily part of the sequence that codes for the final gene product.

[0102] "Translation leader sequence" refers to a DNA sequence located between the promoter sequence and the coding sequence of a gene. Translation leader sequences are present in the fully processed mRNA upstream of the translation start sequence. Translation leader sequences may affect the processing of the primary transcript into mRNA, mRNA stability, or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, GD (1995) Molecular Biotechnology 3:225).

[0103] "3' non-coding sequence" refers to a DNA sequence located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences that code for regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of a pre-mRNA. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., Plant Cell 1:671-680 (1989).

[0104] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. An RNA transcript is called a primary transcript if it is a perfect complementary copy of a DNA sequence, or it may be an RNA sequence derived from post-transcriptional processing of a primary transcript and is called mature RNA. "Messenger RNA (mRNA)" refers to RNA that does not have introns and can be translated into protein by a cell. "cDNA" refers to DNA that is complementary to an mRNA template and is synthesized from an mRNA template using the enzyme reverse transcriptase. cDNA may be single-stranded or may have been converted to double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to an RNA transcript that includes an mRNA and can be translated into protein in a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and blocks expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be to any part of a particular gene transcript, i.e., the 5' non-coding sequence, the 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts and are intended to define the antisense RNA of a message.

[0105] The term "endogenous RNA" refers to any RNA, naturally occurring or non-naturally occurring, i.e., encoded by any nucleic acid sequence present in the genome of the host prior to transformation with a recombinant construct provided herein, whether introduced by recombinant means, mutagenesis, etc.

[0106] The term "non-naturally occurring" means man-made, not corresponding to what is normally found in nature.

[0107] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is regulated by the other. For example, a promoter is operably linked to a coding sequence when it is capable of regulating the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to a regulatory sequence in a sense or antisense orientation. In another example, a complementary RNA region may be operably linked, either directly or indirectly, to the 5' of the target mRNA, or to the 3' of the target mRNA, or within the target mRNA, or the first complementary region is 5' of the target mRNA and its complement is 3' of the target mRNA.

[0108] The term "expression" as used herein refers to the production of a functional end product. Gene expression involves the transcription of the gene and the translation of mRNA into precursor or mature protein. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of a target protein. "Cosuppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

[0109] "Mature" protein refers to a polypeptide that has been post-translationally processed, i.e., the pre- or propeptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA, i.e., the pre- and propeptides are still present. The pre- and propeptides may be, but are not limited to, intracellular localization signals.

[0110] "Stable transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus or DNA-containing organelle of a host organism, resulting in gene expression without integration or stable inheritance. A host organism containing a transformed nucleic acid fragment is called a "transgenic" organism. The term "transformation" as used herein refers to both stable and transient transformation.

[0111] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in more detail in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook").

[0112] The term "recombinant" refers to the artificial combination of two otherwise separated segments of sequence, for example, by chemical synthesis or by the manipulation of isolated segments of nucleic acid by genetic engineering techniques.

[0113] "PCR" or "polymerase chain reaction" is a technique for synthesizing large quantities of specific DNA segments, consisting of a series of repeated cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, double-stranded DNA is heat denatured, two primers complementary to the 3' boundaries of the target segment are annealed at low temperature, and then extended at an intermediate temperature. A set of three successive steps is called a cycle.

[0114] Polymerase chain reaction ("PCR") is a powerful technique used to rapidly amplify DNA millions of times by repeatedly replicating a template. (Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich et al., European Patent Application No. 50,424; European Patent Application No. 84,796; European Patent Application No. 258,017; European Patent Application No. 237,362; Mullis, European Patent Application No. 201,184; Mullis et al., U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194). In this process, a set of specific in vitro synthesized oligonucleotides is used to prime DNA synthesis. The design of the primers depends on the sequence of the DNA to be analyzed. This technique works by melting the template at high temperature, allowing primers to anneal to complementary sequences within the template, and then replicating the template with DNA polymerase over many cycles (usually 20-50).

[0115] The products of the PCR reaction are analyzed by separation on an agarose gel followed by visualization by ethidium bromide staining and UV transillumination. Alternatively, radioactive dNTPs may be added to the PCR to incorporate a label into the product. In this case, the products of the PCR are visualized by exposing the gel to X-ray film. An additional advantage of radioactively labeling the PCR products is that the levels of individual amplification products can be quantified.

[0116] The terms "recombinant construct", "expression construct" and "recombinant expression construct" are used interchangeably herein. These terms refer to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodologies well known to those skilled in the art. Such constructs may be used by themselves or in conjunction with a vector. If a vector is used, the choice of vector will depend on the method that will be used to transform the host plant, as is well known to those skilled in the art. For example, a plasmid may be used. Those skilled in the art will be well aware of the genetic elements that must be present in a vector to successfully transform, select and grow a host cell containing any of the isolated nucleic acid fragments described herein. Those skilled in the art will also recognize that different independent transformation events will result in different expression levels and patterns (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), and therefore multiple events must be screened to obtain a line that exhibits the desired expression levels and patterns. Such screening can be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0117] The terms "isolated" and "purified" as used with respect to a substance (such as an antibody) provided herein indicate that the substance is substantially free of at least one other substance that may be contained in the natural source. Thus, "isolated antibody" and "purified antibody" refer to an antibody that is substantially free of cellular material, such as carbohydrates, lipids, or other contaminating proteins, from the cell or tissue source from which the antibody (protein) is derived. If the antibody is chemically synthesized, the term refers to an antibody that is substantially free of chemical precursor materials or other chemicals. In a preferred embodiment, the antibody of the present invention is isolated or purified.

[0118] Methods for Producing Antibodies The invention also provides a method for producing monoclonal antibodies from non-human, non-mouse animals by immunizing non-human transgenic animals containing human immunoglobulin loci with peptide immunogens as described herein. Such animals can be generated using methods known in the art. In a preferred embodiment, the non-human animal may be a rat, sheep, pig, goat, cow, or horse. Antibody-producing immortalized hybridomas can be prepared from the immunized animals. After immunization, the animals are sacrificed and splenic B cells are fused with immortalized myeloma cells as is well known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lines). After fusion and antibiotic selection, the hybridomas are screened using an antigen (e.g., globulomer) or a portion thereof, or cells expressing the antigen of interest. In a preferred embodiment, initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA. An example of ELISA screening is provided in WO 00 / 37504, which is incorporated herein by reference.

[0119] The antibody-producing hybridomas are selected, cloned, and further screened for desirable characteristics, including robust hybridoma growth, high antibody production, and desirable antibody characteristics, as further discussed below. Hybridomas can be cultured and expanded in vivo in syngeneic animals, animals that lack immune systems, such as nude mice, or in vitro in cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those skilled in the art. Preferably, the immunized animal is a non-human animal that expresses human immunoglobulin genes, and splenic B cells are fused to a myeloma derived from the same species as the non-human animal.

[0120] In one aspect, the present invention provides hybridomas that produce monoclonal antibodies for use in the treatment, diagnosis, and prevention of Alzheimer's disease. In a preferred embodiment, the hybridomas are murine hybridomas. In another preferred embodiment, the hybridomas are produced in non-human, non-mouse species, such as rats, sheep, pigs, goats, cattle, or horses. In another embodiment, the hybridomas are human hybridomas, in which a human non-secretory myeloma is fused with a human cell that expresses an antibody against a globulomer.

[0121] Recombinant antibodies can be produced from single isolated lymphocytes using a procedure known in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Pat. No. 5,627,052, WO 92 / 02551, and Babcock, JS et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7843-7848. In this method, single cells secreting the antibody of interest (e.g., lymphocytes from an immunized animal) are screened using an antigen-specific hemolytic plaque assay in which an antigen (e.g., a globulomer) or a fragment thereof is coupled to sheep red blood cells using a linker such as biotin and used to identify single cells secreting antibodies with specificity for the antigen. After identifying the antibody-secreting cells of interest, heavy and light chain variable region cDNAs can be rescued from the cells by reverse transcriptase PCR, and then these variable regions can be expressed in mammalian host cells, such as COS or CHO cells, in the background of an appropriate immunoglobulin constant region (e.g., human constant region). The host cells transfected with the amplified immunoglobulin sequences derived from the in vivo selected lymphocytes can then be subjected to further analysis and selection in vitro, for example, by panning the transfected cells to isolate cells expressing antibodies against IL-18. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods, such as those described in WO 97 / 29131 and WO 00 / 56772.

[0122] The term "epitope" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen to which an antibody binds. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0123] The term "immunizing" as used herein refers to the process of presenting an antigen to an immune repertoire, whether that repertoire exists in a natural, genetically unmodified organism or in a transgenic organism modified to present an artificial human immune repertoire. Similarly, an "immunogenic preparation" is a formulation of an antigen with an adjuvant or other additive that will enhance the immunogenicity of the antigen. An example would be co-injecting a purified peptide or peptide immunogen into a mouse with complete Freund's adjuvant, in some embodiments, the adjuvant is covalently linked to the peptide. "Hyperimmunization," as defined herein, is the act of presenting an antigen in an immunogenic preparation to a host animal multiple times in succession with the aim of eliciting a strong immune response.

[0124] It should also be noted that the subject invention includes not only the full-length antibodies described above, but also parts or fragments thereof, such as the Fab portions thereof. In addition, the subject invention encompasses any antibody having the same properties as the antibodies of the present invention, e.g., in terms of binding specificity, structure, etc.

[0125] "Antibody" refers to a glycoprotein having the same structural characteristics. Antibodies exhibit binding specificity to a specific antigen. As used herein, "antigen" refers to a protein capable of binding to a corresponding antibody and inducing an antigen-antibody reaction in vivo.

[0126] A protein, the main component of amyloid, is a peptide consisting of 40-42 amino acids, and is known to be produced by the action of proteases from a precursor protein called amyloid precursor protein (APP). In addition to the amyloid fibrils collected in the ultracentrifuged sediment fraction, the amyloid molecules produced from APP include multimeric non-fibrillar assemblies in addition to soluble monomers. "Aβ multimers" refer to non-fibrillar assemblies, which may include, for example, Aβ40 (Aβ1-40) multimers and Aβ42 (Aβ1-42) multimers or combinations thereof. For example, the "Aβ42 multimers" described herein are molecules that exhibit a molecular weight of 45-160 kDa by SDS-PAGE and 22.5-1,035 kDa by Blue Native PAGE. When using molecular sieves, such molecules are mainly collected in the >100 kDa retention solution. When observed by atomic force microscopy, these molecules display a morphology characterized by a mixture of granular, bead-like, and ring-like molecules with heights ranging from 1.5 to 3.1 nm. By gel filtration, molecules with molecular weights of 680 kDa or higher can be eluted in void volume fraction 8, and molecules with molecular weights between 17 and 44 kDa in fraction 15.

[0127] There are no limitations on the origin or form of the antibodies described herein, so long as they bind to Aβ multimers but not Aβ monomers.

[0128] Monoclonal antibodies can be produced using known techniques. For example, monoclonal antibodies can be produced by the hybridoma method first described by Kohler and Milstein (Nature 256: 495-7, 1975) or by recombinant DNA method (Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-7, 1984), but the methods are not limited thereto. For example, when using the hybridoma method, Aβ multimers (e.g., Aβ tetramers described in the Examples) are used as sensitizing antigens, and immunization is carried out according to conventional immunization methods. The obtained immune cells can be fused with known parent cells by conventional cell fusion methods, and monoclonal antibody-producing cells can be screened and isolated using conventional screening methods.

[0129] Monoclonal antibodies can be produced as follows: Balb-c mice are immunized with a peptide immunogen as described herein, optionally emulsified with complete Freund's adjuvant, by injecting the antigen into the footpad. Booster immunizations, for example six times, are then performed. Hybridomas are then produced from the inguinal lymph nodes, for example by fusing with Sp2 / O-Ag14 cells using polyethylene glycol 1500.

[0130] The animal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used in cell fusion. Generally, rodents, lagomorphs, or primates are used. Rodents include, for example, mice, rats, and hamsters. Lagomorphs include, for example, rabbits. Primates include, for example, catarrhine (Old World) monkeys such as Macaca fascicularis, Macaca mulatta, Mantled monkeys, and chimpanzees.

[0131] The animal is immunized with the sensitizing antigen according to known methods, for example, in a standard method, immunization is carried out by intraperitoneally or subcutaneously injecting the sensitizing antigen into the mammal.

[0132] An example of a parent cell to be fused with the above-mentioned immune cells is Sp2 / O-Ag14 cell, which will be described in the Examples below, although various other known cell lines can be used.

[0133] The above-mentioned cell fusion between immune cells and myeloma cells can be carried out basically according to known methods, including, for example, the Kohler and Milstein method (Kohler and Milstein C., Methods Enzymol. (1981) 73, 3-46).

[0134] The hybridomas thus obtained are selected by culturing in a conventional selection medium, such as HAT culture medium, containing hypoxanthine, aminopterin, and thymidine. The culturing in the HAT culture medium referred to above is generally continued for several days to several weeks, a time sufficient to kill cells other than the desired hybridoma (non-fused cells). Next, a conventional limiting dilution method is performed to screen and single-clone hybridomas that produce the desired antibody.

[0135] The hybridoma obtained is then transplanted into the abdominal cavity of a mouse, and ascites containing the desired monoclonal antibody is extracted. For example, the antibody can be purified from the ascites by conventional protein separation and / or purification methods, such as a selected combination of column chromatography, including but not limited to affinity chromatography, filtration, ultrafiltration, salt precipitation, dialysis, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing (Antibodies: A Laboratory manual, Harlow and David, Lane (edit.), Cold Spring Harbor Laboratory, 1988).

[0136] The affinity column can be a Protein A column or a Protein G column. Examples of Protein A columns that can be used include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0137] Chromatography (except affinity chromatography) includes ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography ("Strategies for Protein Purification and Characterization: A Laboratory Course Manual", Daniel R Marshak et al., Cold Spring Harbor Laboratory Press, 1996). When performing chromatography, liquid phase chromatographic methods such as HPLC and FPLC can be used.

[0138] The monoclonal antibody-producing hybridomas thus prepared may be subcultured in a conventional culture medium or may be stored for a long period in liquid nitrogen.

[0139] For antibody production, any mammal can be immunized with the immunogen. However, when preparing monoclonal antibodies by producing hybridomas, the compatibility with the parent cells used in cell fusion for hybridoma production is preferably taken into consideration.

[0140] Generally, rodents, lagomorphs, or primates are used for immunization. Rodents include, for example, mice, rats, and hamsters. Lagomorphs include, for example, rabbits. Primates include, for example, catarrhine (Old World) monkeys, such as Macaca fascicularis, Macaca mulatta, Mantled monkeys, and chimpanzees.

[0141] The use of transgenic animals with human antibody gene repertoires is known in the art (Ishida et al., Cloning and Stem Cells 4: 91-102, 2002). As with other animals, human monoclonal antibodies can be obtained by immunizing the transgenic animals, then harvesting antibody-producing cells from the animals and fusing them with myeloma cells to produce hybridomas, from which anti-protein human antibodies can be prepared (see WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 33735, and WO 96 / 34096).

[0142] Alternatively, lymphocytes immortalized with oncogenes can be used to produce monoclonal antibodies. For example, human lymphocytes infected with EB virus or the like are immunized in vitro with an immunogen. The immunized lymphocytes are then fused with human-derived myeloma cells (such as U266) that can divide indefinitely, thus obtaining hybridomas that produce the desired human antibodies (JP Patent Publication 63-17688 (Unexamined Japanese Patent Application Publication)).

[0143] Once a monoclonal antibody is obtained by any of the above methods, it can also be prepared using genetic engineering methods (see, for example, Borrebaeck CAK and Larrick JW, Therapeutic Monoclonal Antibodies, MacMillan Publishers, UK, 1990). For example, a recombinant antibody can be prepared by cloning DNA encoding a desired antibody from an antigen-producing cell, such as an antibody-producing hybridoma or an immunized lymphocyte, and then inserting the cloned DNA into an appropriate vector and transecting the vector into a suitable host cell. Such recombinant antibodies are also included in the present invention.

[0144] Diagnostic methods, compositions, and kits Despite growing evidence that soluble oAβ plays a central role in early AD pathogenesis, there remains a large unmet need for sensitive and specific methods to non-invasively detect and quantify oAβ, for example in human plasma. The monoclonal antibodies 1G5 and 71A1, generated by using a synthetic Aβ1-40 cyclic peptide as an immunogen, have been extensively characterized and are used to develop the multimer-specific immunoassays described herein. As shown herein, 1) 1G5 and 71A1 recognize soluble oAβ from human brain, CSF, and plasma without showing reactivity to Aβ monomer, 2) 71A1 neutralizes synaptic toxicity of oAβ-rich AD brain extracts in electrophysiological assays, and 3) an ultrasensitive immunoassay using 71A1 as a capturer paired with 3D6 as a detector achieves a lower LLoQ of 0.6 pg / mL and reliably quantifies oAβ in human brain, CSF, and plasma with low coefficient of variation (CV). The availability of a sensitive and specific immunoassay to quantify endogenous human oAβ in blood should enable future studies on the dynamic process of Aβ multimerization and disassembly in the brain and biofluids and establish correlations between oAβ and other known AD biomarkers, including Aβ monomers [32–34], tau fragments [35, 36], and pTau [37–43] in blood. Moreover, these findings support the potential therapeutic benefit of neutralizing 71A1-immunoreactive oAβ. Collectively, these advances will improve our understanding of the pathological role of oAβ in AD and pave the way for monitoring this important pathogenic form of amyloid b protein in Alzheimer's disease and age-related β-amyloidosis.

[0145] Since the detection of synthetic and natural Aβ multimers revised the amyloid hypothesis almost 20 years ago [1, 2], there have been continuous efforts to generate better tools to visualize oAβ and distinguish it from Aβ monomers and fibrils whose structures may partially overlap with smaller multimeric species. Monoclonal antibodies have been developed in an attempt to recognize and even neutralize soluble oAβ, which is believed to be a more synaptic toxic form of Aβ compared to insoluble Aβ plaques in the brain [3, 4, 14]. Among them, monoclonal antibody 1C22

[26] has been shown to be highly specific for both brain and CSF oAβ [30, 53]. We previously quantified 1C22-positive multimers in the CSF of 104 AD subjects enrolled in the ABBY and BLAZE phase 2 clinical trials of Roche's anti-Aβ antibody crenezumab and found that multimeric Aβ levels measured by the 1C22 / 3D6 assay were significantly reduced in a high proportion of crenezumab-treated patients, whereas no systematic changes occurred in the placebo group

[31] . These data prompted us to further test novel antibodies against oAβ with potentially higher avidity and specificity. By designing cyclized Aβ peptide immunogens that could mimic the dimeric conformation, the positive clones 1G5 and 71A1 showed high binding capacity and specificity to the immunogen and synthetic Aβ multimers. In this study, we tested these two novel antibodies for their ability a) to specifically recognize oAβ, b) to quantify oAβ in human brain extracts, CSF, and plasma, and c) to neutralize the synaptic toxicity of diffusible Aβ multimers derived from AD cerebral cortex.

[0146] We first investigated the ability of 1G5 and 71A1 to bind to Aβ assemblies in AD brain soaked extracts. These extracts have been identified to contain oAβ species that are highly diffusible and synaptotoxic

[11] . We found that both 1G5 and 71A1 successfully pulled down Aβ, as investigated by IP immunoblots probed with both mid-region and N-terminal (Asp-1) specific antibodies, and by IP-ELISA using ELISAs to detect Aβx-40 and x-42. Interestingly, compared to the multimer-specific benchmark antibody 1C22

[30] , both 1G5 and 71A1 showed much lower binding capacity to soluble Aβ species in human brain soaked extracts. Similarly, when we compared these three antibodies by immunohistochemistry, 1G5 and 71A1 recognized plaques in unfixed cryostat sections, as confirmed by double labeling with the standard Aβ antibody D54D2. All three multimer-preferring antibodies stained Aβ deposits well in native cryosections, but staining intensity was substantially reduced when sections were fixed with 4% PFA, suggesting that these antibodies recognize conformational epitopes that are sensitive to fixative cross-linking. 71A1 and 1G5 recognized a smaller portion of Aβ deposits in the brain than 1C22. Nevertheless, 71A1 showed a consistent ability to protect against inhibition of hippocampal LTP induced by AD brain soaked extracts, reinforcing previous findings that a small portion (~12%) of brain Aβ obtained by simple diffusion from cortical slices in as little as 30 min confers much of the reversible synaptic toxicity

[11] . Furthermore, 71A1 affinity-purified oAβ from AD soaked extracts conferred potent synaptic toxicity, and 71A1, in turn, can neutralize the toxicity of soluble oAβ from AD brains, indicating attractive potential therapeutic properties of this new antibody.

[0147] In contrast to these data on soluble Aβ from brain (Fig. 1C), our IP-ELISA showed a reversed rank order of Aβ-binding capacity in CSF: 71A1>1G5>>1C22 (Fig. 3A). This difference between brain-soaked extracts and CSF was striking and can be interpreted as suggesting differences in soluble oAβ assembly between brain and CSF. Furthermore, all three of these oAβ-preferring antibodies immunoprecipitated Aβ40 and Aβ42 equally, suggesting that oAβ in CSF contains diverse Aβ monomer variants. To quantify low levels of oAβ from biological fluids such as plasma more accurately than immunoprecipitation, we developed an ultrasensitive sandwich immunoassay on the Erenna SMCxPRO platform (Millipore) using 71A1 as the capture antibody and 3D6 (against the Aβ Asp-1 N-terminus) as the detector antibody, achieving an LLoQ of 0.6 pg / mL, high reproducibility as indicated by low intra-assay CV% (<10%), and excellent (~100%) recovery (Figure 4D). The specificity of the 71A1 / 3D6 immunoassay for oAβ was demonstrated by the strong preference of 71A1 for synthetic ADDLs[1] and S26C Aβ1-40 dimers

[45] over their monomeric components (Figure 4).

[0148] After thus validating the stability and relative multimer specificity of the 71A1 / 3D6 immunoassay for oAβ prepared from synthetic Aβ peptides, we performed the assay on native SEC fractions of AD brain soaked extracts and observed that the assay recognized high MW Aβ multimers and not Aβ monomers (Figures 5C, 5D), similar to the previously reported multimer-specific 1C22 / 3D6 assay

[53] . After confirming the multimer specificity of the 71A1 / 3D6 assay, we proceeded to quantify oAβ levels in human CSF and plasma. Importantly, we found that 71A1-immunoreactive oAβ levels in CSF (n=29) correlated significantly with total tau and pT181 tau in the same samples, as measured by the CLIA-approved ADmark assay. However, we did not observe a correlation between the 71A1 / 3D6 signal and Aβ1-42 monomer levels.

[0149] We then proceeded to characterize the 71A1 / 3D6 immunoassay on human plasma, which was the primary goal of this study. To demonstrate the validity of quantification of oAβ in plasma free from the effects of plasma matrix interference, we investigated 1) dilution recovery, 2) spike recovery with three different natural Aβ sources (human brain soluble homogenate, human brain immersion extract, and CSF), 3) immunodepletion with 71A1, 1C22, and negative control antibody 4-64 (raised against HIV glycoprotein 120), and 4) brain homogenate, CSF, and plasma treated with GnCl, a strong chaotropic salt that rapidly disassembles oAβ into monomers. The 71A1 / 3D6 immunoassay passed all these tests, 1) showing optimal recovery in both plasma dilution and spike-in experiments, 2) showing immunodepleting signal with 71A1 but not with 1C22 or control antibodies, and 3) 71A1 signal was significantly reduced or absent after GnCl treatment in all three sample types.Finally, we performed the 71A1 / 3D6 assay on a plasma cohort of 73 cognitively normal human subjects (demographic data in Table 1) and obtained a mean (+ / -SD) concentration of 43.34±29.09 pg / mL of 71A1 positive multimers in plasma.

[0150] Our study provides several salient findings. First, we report the design and detailed characterization of a new antibody that recognizes a conformational epitope of native human multimeric Aβ. Second, we show that 1G5 and 71A1 have higher binding capacity for oAβ in biofluids than the reference multimer-preferring antibody 1C2. Third, 71A1 neutralizes the synaptic toxicity of highly diffusible multimers in AD brain soaked extracts. Fourth, the novel 71A1 / 3D6 sandwich ELISA sensitively and reproducibly quantifies oAβ in human plasma. To our knowledge, this is the first report of the quantification of oAβ in human plasma using a multimer-selective conformational antibody. This assay will now be a unique tool to investigate the biological features of endogenous oAβ, including its structural properties, its dynamics in human plasma and CSF, and its functional cytotoxicity. We found three reports of oAβ plasma immunoassays [55-57]. All of these involve the use of identical capture and detector antibodies, and therefore quantification requires at least two identical exposed epitopes on the surface. One such assay, using 82E1 as both capture and detector antibodies, is commercially available from Immuno-Biological Laboratories (IBL). Such an assay will recognize a broad range of Aβ, from dimers to protofibrils to fibrils, as long as the N-terminal epitope is available and exposed. Also, all reported assays lack detailed characterization such as dilution linearity, spike-in recovery, and immunodepletion to test assay specificity. The exemplary 71A1 / 3D6 assay described herein exhibits improved specificity by using a novel multimer-specific antibody as a capturer that does not detect monomers, and improved sensitivity with the SMCxPRO system.

[0151] Now that this new assay is established and technically validated, we and other investigators can use it to systematically investigate large, well-defined cross-sectional and longitudinal human cohorts and correlate plasma oAβ levels with other established and emerging AD biomarkers, such as the Aβ monomer assay that detects all six C-terminal variants from Aβ37 to Aβ43

[32] , the NT-1 tau assay that detects N-terminal tau fragments that correlate with multiple AD phenotypes [35, 36], and the very promising recent assay that detects tau phosphorylated at Thr181, Thr217, and Thr231 (see Introduction). Such detailed correlational analyses in multiple cohorts, including monitoring the effects of advanced anti-amyloid treatments in CSF and plasma, should reveal further insights into the involvement of oAβ dynamics in the early pathogenesis and course of AD. The generation, analytical validation, and initial application of this sensitive and specific assay for endogenous oAβ in human plasma provides a long-sought method to detect and track a species that extensive evidence from multiple laboratories suggests is the key bioactive form of Aβ in AD and therefore a primary target for disease-modifying therapies.

[0152] Thus, provided herein is a method for assaying oAβ present in a sample comprising one or more biological fluids, for example from a human subject, or an animal model, for example a humanized APP mouse, or a veterinary subject, for example a cat or a dog, as described herein. Such a method can be used to quantitatively assay oAβ in a sample. Such a method can be used on a fluid sample from a human patient, for example a sample comprising blood or plasma. The sample can be obtained using a method known in the art. The sample can include, for example, tissue, whole blood, plasma, serum, urine, saliva, exhaled air, exosomes or exosome-like microvesicles, lymph, cerebrospinal fluid, or sputum.

[0153] In some embodiments, a sandwich immunoassay is used that uses a capture antibody bound to a surface (e.g., a surface such as a surface of a plate or bead) and a labeled detection antibody. Such methods may include a step of capturing oAβ present in a sample with a first agent or probe (capture antibody) that recognizes and binds to oAβ in the sample. Preferably, the capture antibody is the 71A1 antibody or 1G5 antibody or an antigen-binding fragment thereof described herein. The capture antibody is preferably attached to a surface, e.g., the surface of a plate such as a multi-well plate, a bead, e.g., a bead, particle, microsphere, nanotube, polymer, plate, disk, or dipstick. The beads may include magnetic beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, e.g., SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, or TEFLON® beads. In some embodiments, the capture antibody is conjugated to a biotin moiety, the surface is coated with streptavidin, and the antibody is attached to the surface via a biotin-streptavidin bond.

[0154] The sample is contacted with a second detection antibody that binds to the oAβ-capture antibody complex. For example, the detection antibody can bind to oAβ, but recognizes a different epitope of the target analyte and does not compete with the capture (e.g., 71A1 or 1G5) antibody for binding to oAβ. Exemplary detection antibodies that bind to oAβ include 3D6 (e.g., Feinberg et al. Alzheimers Res Ther. 2014;6:31) or 82E1, and 71A1 or 1G5 can also be used as a detector in a sandwich assay. Alternatively, the capture antibody can include an affinity tag, and the detection antibody can bind to the affinity tag. Examples of affinity tags include hemagglutinin (HA), HIS tag, FLAG tag (DYKDDDDK, SEQ ID NO: 38), glutathione S-transferase (GST), green fluorescent protein (GFP), S tag, Strep tag, VSVG, V5, myelin basic protein (MBP), c-myc, and combinations thereof (see, for example, Kimple et al., Curr Protoc Protein Sci. 2013; 73: Unit-9.9). Detection antibodies that bind to tags are known and are commercially available, for example, from BioRad, ThermoFisher, and other vendors. Preferably, the detection antibody is labeled with, for example, a radioisotope, a chemiluminescent substance, a fluorescent substance, a metal complex, a bioluminescent substance such as luciferase, or a nucleic acid.

[0155] Such methods further include a step of detecting and optionally quantifying the concentration of oAβ in the sample, for example, by using a radioimmunoassay (RIA) when the detection antibody is labeled with a radioisotope, a chemiluminescent immunoassay (CIA) when the detection antibody is labeled with a chemiluminescent substance, a fluorescent immunoassay (FIA) when the detection antibody is labeled with a fluorescent substance, an electrochemiluminescent immunoassay (ECLIA) when the detection antibody is labeled with a metal complex, a bioluminescent immunoassay (BLIA) when the detection antibody is labeled with a bioluminescent substance such as luciferase, an immuno-PCR method including amplifying an antibody-labeled nucleic acid by PCR and detecting the nucleic acid when the detection antibody is labeled with a nucleic acid, a turbidimetric immunoassay (TAI) that detects turbidity caused by the formation of an immune complex, a latex agglutination turbidimetric assay (LA) that detects latex aggregated by the formation of an immune complex, or an immunochromatographic assay using a reaction on a cellulose membrane. In some embodiments, the method is a fluorescence-based ELISA platform such as SMCxPRO or Erenna (MilliporeSigma).

[0156] Detection can be facilitated by coupling the antibody, or an antigen-binding fragment, variant, or derivative thereof, to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals used in various positron emission tomography methods, and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see, for example, U.S. Pat. No. 4,741,900. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent substances include luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include: 125 I, 131 I, 111 In, or 99In some embodiments, the fluorescent label is a bright near-infrared fluorescent dye, ideally with suitable excitation for 594 nm or 633 nm laser lines. In some embodiments, the label is or comprises a fluorescent protein. Many reporter proteins are known in the art, including Green Fluorescent Protein (GFP), variant of Green Fluorescent Protein (GFP10), enhanced GFP (eGFP), TurboGFP, GFPS65T, TagGFP2, mUKGEmerald GFP, superfolder GFP, GFPuv, destabilized EGFP (dEGFP), Azami Green, mWasabi, Clover, mClover3, mNeonGreen, NowGFP, Sapphire, T-Sapphire, mAmetrine, photoactivatable GFP (PA-GFP), Kaede, Kikume, mKikGR, tdEos, Dendra2, mEosFP2, Dronpa, blue fluorescent protein (BFP), eBFP2, Azurite BFP, mTagBFP, mKalamal, mTagBFP2, shBFP, cyan fluorescent protein (CFP), eCFP, Cerulian.CFP, SCFP3A, destabilized ECFP (dECFP), CyPet, mTurquoise, mTurquoise2, mTFPI, photoswitchable CFP2 (PS-CFP2), TagCFP, mTFP1, mMidriishi-Cyan, aquamarine, mKeima, mBeRFP, LSS-mKate2, LSS-mKatel, LSSmOrange, CyOFP1, Sandercyanin, red fluorescent protein (RFP), eRFP, mRaspberry, mRuby, mApple, mCardinal, mStable, mMaroonl, mGarnet2, tdTomato, mTanger ine, mStrawberry, TagRFP, TagRFP657, TagRFP675, mKate2, HcRed, t-HcRed, HcRed-Tandem, mPlum, mNeptune, NirFP, Kindling, Near-infrared fluorescent protein, Yellow fluorescent protein (YFP), eYFP, Destabilized EYFP (dEYFP), TagYFP, Topaz, Venus, SYFP2, mCherry, PA-mCherry, Citrine, mCitrine, Ypet, IANRFP-AS83, mPapayal, mCyRFP1, mHoneydew, mBanana, mOrange, Kusabira Orange, Kusabira Orange2, mKusabira Orange, mOrange2, mKOK, mKO2, mGrapel, mGrape2, zsYellow, eqFP611, Sirius, Sandercyanin, shBFP-N158S / L173I , near-infrared protein, iFP1.4, iRFP713, iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, miRFP670, BrilliantViolet (BV) 421, BV605, BV510, BV711, BV786, PerCP, PerCP / Cy5.5, DsRed, DsRed2, mRFP1, pocilloporin, Renilla GFP, MonsterGFP, paGFP, or Phycobiliprotein, or a biologically active variant or fragment of any one thereof. Other proteins, such as luciferase, may also be used.

[0157] The antibody, or its antigen-binding fragment, variant, or derivative, can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0158] One way an antibody, or antigen-binding fragment, variant, or derivative thereof, can be detectably labeled is by linking it to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol. 31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme bound to the antibody will react with a suitable substrate, preferably a chromogenic substrate, to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric, or visual means.Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.In addition, detection can be achieved by colorimetric methods using a chromogenic substrate for the enzyme.Detection can also be achieved by visually comparing the extent of enzymatic reaction of the substrate with a similarly prepared standard.

[0159] Detection can also be achieved using any of a variety of other immunoassays. For example, radioactive labeling of antibody or its antigen-binding fragment, variant or derivative allows detection of the antibody using radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated herein by reference). Radioisotopes can be detected by means including, but not limited to, gamma counters, scintillation counters or autoradiography.

[0160] Antibodies, or antigen-binding fragments, variants, or derivatives thereof, can also be detectably labeled using fluorescence-emitting metals, such as 152Eu or others of the lanthanide series. Such metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0161] Techniques for conjugating various moieties to antibodies, or antigen-binding fragments, variants, or derivatives thereof, are well known. For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer See, for example, "Monoclonal Antibodies For Cancer Detection And Therapy", Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62 (1982), 119-158.

[0162] The present disclosure includes methods for diagnosing a subject as having amyloidosis, such as CAA or Alzheimer's disease (AD). Such methods include obtaining a sample from a subject, and evaluating the presence and / or level of oAβ in the sample, and comparing the presence and / or level with one or more references, such as a control reference representing a normal level of oAβ, such as the level of an unaffected subject, and / or a disease reference representing the level of a protein associated with amyloidosis (e.g., CAA or AD), such as the level in a subject with AD or amyloidosis. In some embodiments, such methods may further include administering a treatment, such as a treatment known in the art or described herein, to the subject if the subject is identified as having amyloidosis, such as AD or CAA. Treatments include cholinesterase inhibitors, such as donepezil, rivastigmine, galantamine; NMDA antagonists, such as memantine; or monoclonal antibodies, such as aducanumab, or antibodies described herein. Alternatively or in addition, such methods may further include recommending or performing further diagnostic testing on the subject, such as mental status and neuropsychological testing, or imaging, such as using a magnetic resonance imaging (MRI) or computed tomography (CT) or positron emission tomography (PET) scan, such as fluorodeoxyglucose (FDG) PET imaging, amyloid PET imaging, or tau PET imaging.

[0163] As used herein, the term "sample" when referring to a material to be tested for the presence of a biological marker using the method of the present invention includes, inter alia, tissue, whole blood, plasma, serum, urine, saliva, exhaled air, exosomes or exosome-like microvesicles (US Pat. No. 8,901,284), lymph, cerebrospinal fluid, or sputum. The type of sample used may vary depending on the identity of the biological marker to be tested and the clinical context in which the method is used.

[0164] The presence and / or levels of oAβ can be assessed using methods known in the art, for example, using quantitative protein immunoassays, including, but not limited to, Western blots; enzyme-linked immunosorbent assays (ELISAs), such as sandwich ELISAs; biotin / avidin type assays; protein array detection; radioimmunoassays; immunohistochemistry (IHC); immunoprecipitation assays; FACS (fluorescence activated cell sorting); mass spectrometry (Kim (2010) Am J Clin Pathol 134:157-162; Yasun (2012) Anal Chem 84(14):6008-6015; Brody (2010) Expert Rev Mol Diagn 10(8):1013-1022; Philips (2014) PLOS One 9(3):e90226; Pfaffe (2011) Clin Chem 57(5): 675-687). Such methods typically include revealing a label, such as a fluorescent, chemiluminescent, radioactive, and enzymatic or dye molecule that provides a signal either directly or indirectly. As used herein, the term "labeling" refers to the coupling (i.e., physical linkage) of a detectable substance, such as a radioactive agent or fluorophore (e.g., phycoerythrin (PE) or indocyanine (Cy5)) to an antibody or probe, as well as to indirect labeling of a probe or antibody by reactivity with a detectable substance (e.g., horseradish peroxidase, HRP).

[0165] In some embodiments, ELISA methods can be used in which a surface, such as a bead or a well of a microtiter plate, is coated with a capture antibody against which the protein is to be tested. A sample containing or suspected to contain a biological marker is then applied to the well. After a sufficient time for antibody-antigen complexes to form, the plate is washed to remove any unbound moieties, and detectably labeled molecules are added. After a sufficient incubation period, the plate is washed again to remove any excess unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. Variations of ELISA methods can also be used, such as competitive ELISA or competitive assays, and sandwich ELISA. Such variations are well known to those skilled in the art.

[0166] In some embodiments, the presence and / or level of oAβ is equivalent to the presence and / or level of the protein in the disease reference, and the subject has one or more symptoms associated with amyloidosis, e.g., AD or CAA. In that case, the subject has or can be diagnosed as having amyloidosis, e.g., AD or CAA. In some embodiments, the subject does not have obvious signs or symptoms of amyloidosis, e.g., AD or CAA, but the presence and / or level of one or more of the assessed proteins is equivalent to the presence and / or level of such protein in the disease reference. In that case, the subject has an increased risk (above the risk level of the general population) of developing amyloidosis, e.g., AD or CAA. In some embodiments, once an individual is determined to have amyloidosis, e.g., AD or CAA, or to have an increased risk of developing amyloidosis, e.g., AD or CAA, then a treatment, e.g., as known in the art or described herein, may be administered.

[0167] Suitable reference value can be determined using methods known in the art, for example, using standard clinical trial methodology and statistical analysis.Reference value can have any relevant form.In some cases, reference comprises a predetermined value for the meaningful level of oAβ, for example, a control reference level representing normal oAβ level, for example, the level in a subject who is not affected or does not have the risk of developing the disease described herein, and / or a disease reference representing the level of a protein associated with a condition associated with amyloidosis, for example, AD or CAA, for example, the level in a subject who has amyloidosis, for example, AD or CAA.

[0168] The predetermined level may be a single cut-off (threshold) value, such as a median or mean, or may be a level that defines an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from other segments. The predetermined level may also be a range of cut-off values ​​(or thresholds), such as a confidence interval. The predetermined level may be established based on a comparison group, such as an association between the risk of developing or the presence of disease in one defined group being 1-fold higher or lower (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold, or more) than the risk or presence of disease in another defined group. The predetermined level may be, for example, a range in which a population of subjects (e.g., control subjects) is evenly (or unequally) divided into groups such as low, medium, and high risk groups, or into quartiles, with the lowest quartile being the subjects with the lowest risk and the highest quartile being the subjects with the highest risk, or into n quantiles (i.e., n equally spaced intervals), with the lowest quartile of the n quantile being the subjects with the lowest risk and the highest quartile of the n quantile being the subjects with the highest risk.

[0169] In some embodiments, the predetermined level is the level or occurrence in the same subject, eg, at a different time point, eg, an earlier time point.

[0170] A subject associated with a given value is typically referred to as a reference subject. For example, in some embodiments, a control reference subject does not have a disorder described herein (e.g., amyloidosis, such as AD or CAA).

[0171] A disease reference subject is a subject who has (or has an increased risk of developing) one or more of amyloidosis, such as AD or CAA. Increased risk is defined as a risk greater than the subject's risk in the general population.

[0172] Thus, in some cases, a level of oAβ in a subject that is less than or equal to a reference level of oAβ indicates a clinical condition (e.g., indicates the absence of a disorder described herein, such as amyloidosis, e.g., AD or CAA). In other cases, a level of oAβ in a subject that is greater than or equal to a reference level indicates the presence of amyloidosis, e.g., AD or CAA, or an increased risk of disease. In some embodiments, the amount by which the level in a subject is less than the reference level is sufficient to distinguish the subject from a control subject, and optionally is statistically significantly lower than the level in the control subject. When the level of oAβ in a subject is equal to a reference level of oAβ, "equal" refers to being approximately equal (e.g., not statistically different).

[0173] The predetermined value may depend on the particular population of subjects (e.g., human subjects) selected. For example, a seemingly healthy population will have a "normal" range of levels of αAβ that is different from the levels that a population of subjects who have, are more likely to have, or are at higher risk of having a disorder described herein will have. Thus, the predetermined value selected may take into account the category (e.g., sex, age, health status, risk, presence of other diseases) to which the subject (e.g., human subject) belongs. Appropriate ranges and categories can be selected with no more than routine experimentation by those skilled in the art.

[0174] In characterizing the likelihood or risk, a number of predetermined values ​​can be established.

[0175] Also provided herein is a kit for quantifying the amount of oAβ in a sample, the kit comprising a capture antibody and a detector antibody as described herein, optionally a positive control, and optionally instructions for using said reagents to quantify oAβ in a sample.

[0176] Therapeutic Methods and Compositions Amyloidosis is a clinical disorder caused by extracellular and / or intracellular deposition of pathogenic amyloid in various tissues. Signs and symptoms of amyloidosis include: severe fatigue and weakness; shortness of breath; numbness, tingling, or pain in the hands and feet; swelling of the ankles and legs; diarrhea or constipation, possibly with bleeding; an enlarged tongue, which may appear wavy at the edges; skin changes such as thickening or easy bruising; and purpura around the eyes. Brain accumulation of amyloid beta peptide (Aβ) in the form of plaques in both the cerebral cortex and blood vessels is associated with cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD).

[0177] There is strong evidence that soluble multimers of amyloid beta protein (oAβ) help initiate the pathogenic cascade of Alzheimer's disease (AD), suggesting therapeutic strategies that target oAβ rather than monomeric or fibrillar Aβ. The antibody 71A1 described herein is approximately 100-fold more sensitive to oAβ than synthetic monomers. Substances that 71A1 specifically immunoprecipitates from AD soluble brain extracts impair synaptic function similarly to intact extracts. As shown herein, preincubating brain extracts with 71A1 neutralizes its synaptic toxicity. The intrinsic activity of 71A1 against disease-associated oAβ makes it therapeutically useful for treating amyloidosis, such as AD or CAA.

[0178] Thus, provided herein is a method for treating a disorder associated with the accumulation of oAβ in a subject, such as amyloidosis, such as AD or CAA. Such a method includes administering a therapeutically effective amount of a 71A1 antibody or an antigen-binding fragment thereof to a subject, such as to the brain of a subject. As used in this context, "treat" means improving at least one symptom of amyloidosis, such as AD or CAA. In many cases, amyloidosis leads to impaired function of the affected organ. Thus, treatment can reduce oAβ and restore or approach normal function. For example, AD and CAA are associated with progressive cognitive decline. Administration of a therapeutically effective amount of a compound described herein for treating AD or CAA will result in a reduction in cognitive impairment, a slowing down of the rate of progressive cognitive decline, or a halt to progressive cognitive decline.

[0179] Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of pharmaceutical compositions comprising or consisting of an antibody described herein, such as mAb 71A1 or 1G5, or an antigen-binding fragment thereof, as an active ingredient. In some embodiments, the antibody is a monoclonal antibody, a non-human animal antibody, a humanized antibody, a chimeric antibody, a human antibody, a minibody, a bispecific antibody, an amino acid sequence modified antibody, a modified antibody conjugated to another molecule (e.g., a polymer such as polyethylene glycol), or a glycomodified antibody comprising the HC CDRs and LC CDRs of mAb 71A1 or 1G5, or the entire HC and LC of mAb 71A1 or 1G5.

[0180] Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration.

[0181] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration, which includes parenteral, e.g., intravenous delivery; intraventricular, intracerebral, or intrathecal injection; or injection into the CSF.

[0182] Methods for formulating suitable pharmaceutical compositions are known in the art.See, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can include the following components: sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose.pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0183] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0184] Sterile injection solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or combination of the components listed above, and then sterilizing by filtration as necessary.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from those listed above.For the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying, thereby obtaining the powder of active ingredient + any additional desired components from the solution that has been previously sterilized and filtered.

[0185] In some embodiments, the therapeutic compound is prepared with a carrier that will protect the therapeutic compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.The compound can also be delivered using, for example, a pump, such as a surgically implanted reservoir pump.Biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Such formulations can be prepared using standard techniques or can be obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Nanoparticles (e.g., liposomes, polymeric nanoparticles, dendrimers, clathrin nanoparticles, or metal nanoparticles), engineered bispecific antibodies (e.g., those that also bind to the transferrin receptor (TfR), insulin receptor (IR), or low-density lipoprotein receptor-related protein-1 (LRP-1) (see, e.g., Faresjo et al., Fluids Barriers CNS. 2021;18:26; Bajracharya et al., Pharmaceutics. 2021 Dec; 13(12): 2014), a combination of focused ultrasound with microbubbles, and extracellular vesicles with blood-brain barrier (BBB) ​​crossing properties can be used to enhance delivery across the BBB. See, e.g., Bajracharya et al., Pharmaceutics. 2021 Dec; 13(12): 2014. Nanoparticles, including liposomes, can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. Alternatively, antibody-encoding viral vectors (such as AAV) can be delivered, for example, containing nucleic acid encoding a therapeutic antibody as described herein (preferably codon-optimized for human use).

[0186] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0187] Dosage An "effective amount" is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is an amount necessary to prevent the onset of a disease or disease symptoms. An effective amount can be administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., effective dosage) of a therapeutic compound will depend on the therapeutic compound selected. The composition can be administered from one or more times daily to one or more times weekly, including every other day. Those skilled in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound as described herein can include a single treatment or a series of treatments.

[0188] The dosage, toxicity, and therapeutic effect of therapeutic compounds can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, for example, to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but care must be taken to design a delivery system that targets such compounds to affected tissue sites in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0189] Data obtained from cell culture assays and animal studies can be used in formulating a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within that range depending on the dosage form used and the route of administration used. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. EXAMPLES

[0190] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0191] [Example 1] Peptide Immunogens The human amyloid b protein 42 (Aβ42) amino acid sequence and the region within the sequence (aa 9-18) used to generate immunogenic peptides are as follows:

[0192] [ka]

[0193] This sequence was chosen because it contains a cluster of amino acids with complex side chains that, when aligned into dimers, trimers, or even more complex structures, may reassociate into unique orientations that would create characteristic three-dimensional structures similar to those that may form early in Aβ aggregation. These structures may represent "neoepitopes" for antibody binding that are not present in the unaggregated monomeric sequence. The main goal of immunogen design was to create a molecule that is ideally a stable dimer, but could also be a stable trimer or even a stable tetramer structure, that would focus the immune response as much as possible on conformational neoantibody epitopes that are created when Aβ initially aggregates and then lost as larger aggregates form.

[0194] A synthetic peptide version of this region, having the sequence CGKCGYEVHHQKLVPNVLKQHHVEYGCK (SEQ ID NO:2), was made for use as an immunogen in Balb / c mice.

[0195] A schematic of the cyclized peptide is shown in Figure 1. Mabs 71A1 and 1G5 were both isolated using this immunogen conjugated to a proprietary T cell epitope peptide.

[0196] This immunogen, also referred to herein as NETL-1, was derived from a stretch of 10 amino acids within the Aβ sequence depicted above. This region was derived from a sequence thought to potentially be involved in the initial conversion from an alpha-helical to a β-sheet conformation, similar to the β-sheet conversion required for the formation of Aβ protofibril aggregates. Allowing the peptide to cyclize before cleavage from the resin would result in the formation of a kink, so the 10 amino acid stretch was repeated in the reverse direction after the addition of a proline and an asparagine. The fully synthesized and cleaved peptide from the resin was conjugated using an N-terminal cysteine ​​to a carrier protein such as sulfhydryl-reactive ELISA plates and maleimide-activated KLH (keyhole limpet hemocyanin) or to a proprietary maleimide-peptide carrier termed Tepi2. The maleimide is sulfhydryl-reactive and the cysteine ​​has a free sulfhydryl group available for conjugation with other sulfhydryl groups or maleimides. An additional lysine was added to the N-terminus of the peptide to increase water solubility. Control peptides for screening included both full-length Aβ40 with a C-terminal cysteine ​​(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV, SEQ ID NO: 40) and the NETL-2 peptide, which is the same Aβ10 amino acid peptide sequence used in NETL1 but does not have the inverted repeat and has an additional N-terminal lysine and cysteine.

[0197] Tepi2 is a sequence that, when presented in the context of class II MHC antigens on antigen-presenting cells (APCs), activates helper T cells to mount an antibody response against the Abeta sequence structure. An exemplary Tepi2 is as follows:

[0198] [ka]

[0199] This is a peptide sequence (SEQ ID NO: 4) that has Pam3Cys (N-α-palmitoyl-S-2,3-bis(palmitoyloxy)-(2RS)-propyl-L-cysteine ​​with palmitoyl-Cys ((RS)-2,3) attached to the second lysine (K4). Pam3Cys is a lipopeptide adjuvant that is known to stimulate the innate immune response. The innate immune response is a response that uses germline-encoded pattern recognition receptors (PRRs) found on many microorganisms or parasites that bind to moieties similar to Pam3Cys. Induction of the innate immune system results in the production of many inflammatory cytokines that can immediately trigger a host defense response to prime an antigen-specific adaptive immune response, such as the induction of specific antibody production. (Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol (2002) 20:197-216).

[0200] The purpose of conjugating Tepi2 to peptide immunogens rather than continuing with large protein carriers is twofold. First, the large protein carriers themselves induce antibody responses, and many of the activated B cells respond to unwanted and irrelevant epitopes found on these proteins. Thus, attaching the Tepi-2 carrier to the subsequent booster limited the continued activation of primed B cells specific for the desired Abeta peptide immunogen. Second, we were unable to measure any antibody responses against the additional amino acid composition of Tepi2 by ELISA using Tepi2-coated plates.

[0201] [Example 2] Biochemistry and Immunology NETL-1 contained three cysteines with sulfhydryls available for conjugation and post-synthetic modification prior to cleavage from the resin (Anaspec, Inc) used to generate the peptide. The peptides were synthesized using FMOC chemistry on an automated peptide synthesizer such as the Applied Biosystems 432A. The peptides have the necessary side chain protecting groups (Trt-tert-butyloxycarbonyl or Boc) throughout the synthesis process to ensure amino acid integrity. These groups must be removed upon cleavage of the final product from the resin used to synthesize the peptide. NETL-1 has two cysteines (C4 and C27) inserted at both c-termini of the peptide. These cysteines were used to cyclize the peptide on-resin prior to final cleavage from the resin. To accomplish this task, the cysteines were protected with acetamidomethyl (ACM) groups. When the ACM groups were removed using a thallium III procedure, these cysteines were preferentially cleaved without affecting the Boc protecting group, which was removed under the different conditions used to cleave the peptide from the resin. Removal of the ACM protecting group generates free sulfhydryls that are believed to preferentially react with each other to form loops on the resin. It is possible that some interpeptide dimerization will also result. Of course, the scheme of creating stable dimeric structures using these synthetic acrobatics to generate potential β-sheet structures does not eliminate any binding preference. Adding amino acids after the C-terminal valine of the initial Abeta 9-18 sequence, or to either the N- or C-terminus of the immunogen, can achieve the same goal of forming stable compact Abeta structures that form early in aggregation. The isolated 71A1 and 1G5 antibodies that respond to tertiary structures rather than the native continuous "linear" sequence were then drawn from a repertoire of responses to many different configurations. Final cleavage from the resin likely results in a stably associated dimer with a free sulfhydryl available at the N-terminus of the peptide for conjugation.

[0202] There are additional amino acid protecting groups within the peptide sequence that can be used to create stable dimerization. For example, the monomethoxytrityl (MMT) protecting group is a versatile amino protecting group that is more sensitive to strong acids, bases, and nucleophiles than the ACM protecting group and therefore easier to remove. The stretch of 10 amino acids used in NETL-1 can also be repeated in tandem, separated by prolines and asparagines, as illustrated below. CGKCGYEVHHQKLVPNGYEVH HQKLVCK ​​(SEQ ID NO: 3)

[0203] The first cysteine ​​(C1) can be protected with a TRT group, which is used for all amino acids during peptide synthesis and cleavage from the resin. For example, the second cysteine ​​(C4) can be protected with an ACT group and the third cysteine ​​(C27) with an MMT group. The MMT group is first removed to allow disulfide bonding between adjacent peptides through a C4 disulfide bridge, and then the ACT group is removed to allow an additional C27 disulfide bridge between the same adjacent peptides. Cleavage of the peptide from the resin can generate a stable peptide structure with adjacent repeating dimers.

[0204] [ka]

[0205] Other protecting groups and strategies can be used with the same goal in this process and in the NETL-1 peptide synthesis process, with the aim of creating stable dimers, trimers, or tetrapeptide aggregates with unique three-dimensional structures not available in the monomeric Aβ40 or Aβ42 peptides.

[0206] A normal immune response involves the activation of both B and T cell subsets of lymphocytes. Responses mounted against complex proteins require the uptake and digestion of peptide fragments derived from that protein followed by cell surface presentation. These fragments are presented in the intracellular environment in association with cell surface expressed molecules called MHC molecules on B cells and specialized cells called APCs or antigen presenting cells. T cells have specialized receptors that recognize these peptide fragments associated with MHC. This recognition initiates intercellular events within the T cell that result in the production and synthesis of cytokines. These cytokines stimulate B cell maturation and ultimately the development of antibody-secreting B cells.

[0207] There are many studies that attempt to understand and elucidate the process and mechanisms of T cell activation. Sequences within proteins that are involved in T cell activation are called T cell epitopes, and the identification of such sequences and the characteristics between such sequences are an area of ​​interest for immunologists. Usually, when an antibody response against a small peptide is desired, the peptide is conjugated to a large carrier molecule such as KLH or BSA. This conjugation process allows B cells and APCs to internalize, process, and present the T cell epitope peptides derived from the large carrier protein. However, the problem with such carriers is that they also have many B cell epitopes, and in most cases, the antibody response against these regions overwhelms the response against the peptide conjugated to the carrier. As a result, over time, the specific titer against the peptide of interest decreases. This phenomenon does not occur when using T cell epitopes, because the only B cell epitopes available for recognition are those derived from the peptide or molecule conjugated to the carrier.

[0208] The T cell epitopes used to stimulate Mab 71A1 and 1G5 are sequences that we have used to initiate mouse antibody responses, particularly against small molecules and peptides.For example, these sequences conjugated to small peptide immunogens that themselves are derived from proteins of interest will generate antibody responses in mice against peptides of interest. No antibody response against T cell epitopes is observed.In addition, we have previously conducted studies showing that only these sequences will lead to T cell proliferation.In this sense, these T cell epitopes will likely present a complex that is recognized by T cells in association with MHC on B cells and APCs.This recognition completes the initiation requirement that will lead to an antibody response against molecules conjugated to these T cell epitopes.

[0209] To reduce any disulfide bonds between NETL-1 peptides, NETL-1 peptides were conjugated to T cell epitope peptides at a 1:1 molar ratio in 0.1 M sodium phosphate buffer pH 6.5 containing 5 mM EDTA. Both controls Aβ42-cysteine ​​and NETL-2 were treated similarly.

[0210] The complete cyclization and cleavage protocol is as follows: First, determine the mass of peptide-resin to be cyclized and then determine the mass of thallium III required for cyclization: (X mM NETL12 x 2) x 543.2 mg / mM = Y mg thallium III. Swell the resin in a glass vial using DMF. Transfer the required volume of thallium III (the same amount added to swell the peptide-resin) to the peptide-resin. Allow the reaction to proceed with stirring for 2 hours at 0°C. Transfer the reaction mixture to an appropriately sized glass sintered filter flask for filtration. Wash the peptide-resin mixture three times with DMF, then wash the peptide-resin mixture three times with DCM.

[0211] The sintered flask is covered and the mixture is allowed to dry overnight. The dried peptide-resin is transferred to a covered glass vial and the yield is determined.

[0212] The next day, the cyclized peptide-resin is weighed and placed into an appropriately sized glass vial containing a magnetic flea or stir bar.

[0213] Prepare a cleavage cocktail containing thioanisole, deionized water, ethanedithiol, and trifluoroacetic acid and chill on ice. Slowly add the entire volume of the chilled cocktail to the vial containing the peptide-resin and stir on ice for 15 min, then at room temperature for 165 min.

[0214] Chill MTBE on ice during the cleavage reaction. Use approximately 35 ml chilled MTBE for every 100-150 mg peptide-resin. Load peptide-resin-cocktail mixture onto Quik-Sep column into chilled MTBE. Wash with TFA. Centrifuge to pellet cleaved peptide, then wash / resuspension peptide pellet with chilled MTBE. Repeat centrifugation and washing 3 times.

[0215] Centrifuge as before to pellet the peptide and dry briefly. Dissolve the pellet in a minimum volume of glacial acetic acid. Repeat freezing and lyophilization one or two times.

[0216] [Example 3] Immunization and antibody screening Balb / c mice were given primary and secondary immunizations with KLH-conjugated NETL-1 peptides. Subsequent boosts were performed at 3-week intervals with T cell epitope-conjugated peptides. This approach allowed KLH to establish a strong immune response to the peptide early on. Before the antibody response to KLH begins to overwhelm the response to the peptide, the T cell epitope allows for further boosting against the structure of interest without further induction of irrelevant antibody responses due to the B cell epitopes contained within KLH. The T cell epitope induces minimal or no B cell responses, but induces a strong T cell proliferation response that is important for B cell induction and maturation.

[0217] The rationale behind the NETL-1 immunogen was that the more focused epitopes formed by the cyclized peptides could restrict the response to a dimeric structure derived from the selected 10aa sequence such that there would be minimal or no induction of a response to the monomeric 10aa peptide sequence found within full-length Aβ40. Both responses were observed. We can partially explain this by the fact that no matter how small the peptide structure, there may always be a minimal epitope available for recognition that cross-reacts with the monomeric sequence. Several monoclonal antibodies specific for both NETL-1 and NETL-2 were isolated. Two isolates, 7a and 1g, were identified that bound to NETL-1 but not to NETL-2 or Aβ40. These isolates were subcloned for further characterization. Two isolated subclones, Mab 71A1 and 1G5, were selected, grown in tissue culture, and purified for confirmation of specificity and future studies. Both 71A1 and 1G5 monoclonal antibodies bound to the cyclized peptide NETL-1, but not to the control wells, the Aβ40 monomer peptide, or the control peptide NETL-2 (Figure 2). Figure 2 shows a standard indirect enzyme-linked immunosorbent assay (ELISA) with purified monoclonal antibodies assayed for binding to Aβ40, NETL1, or NETL2 peptides conjugated to maleimide-activated (SH-reactive) microtiter plates. After 1 hour of incubation, the wells were washed and then 100 μl of horseradish peroxidase (HRPO)-conjugated goat anti-mouse secondary antibody was added, and the plates were incubated for an additional hour before the addition of TMB (3,3',5,5'-tetramethylbenzidine) substrate. The absorbance was measured at 650 nm in a spectrophotometer.

[0218] [Example 4] An ultrasensitive immunoassay for detecting and quantifying soluble Aβ multimers in human plasma In this example, a sensitive, high-throughput and inexpensive method is described to analyze large cohorts of aged and AD subjects to quantify synaptotoxic oAβ in human plasma to assess the dynamics of this important pathogenic species and its response to therapy.

[0219] method In Example 4, the following method was used.

[0220] Generation of Aβ multimer-specific antibodies 71A1 and 1G5 Monoclonal antibodies 71A1 (a subclone of the parent clone 7A1a) and 1G5 were raised against synthetic conformational peptide immunogens designed to potentially mimic the three-dimensional structure formed upon dimerization of monomeric Aβ. 1-40 A peptide immunogen comprising amino acid residues 9-18 of was synthesized, cyclized, and post-synthetically modified to enable folding into a stable dimer former (patent pending). The rationale behind selecting residues 9-18 of the Aβ peptide was that the amino acids in this region were deemed more likely to reassociate into a unique three-dimensional structure that is present only in the aggregated form of Aβ. Balb / c mice were immunized with this conformational peptide immunogen prior to splenectomy procedure using a maleimide-activated keyhole limpet hemocyanin (KLH) carrier complex for primary and secondary immunizations, and a proprietary helper 2 T cell epitope for subsequent boosts. Fusions were performed using immunized mouse splenocytes and mouse myeloma F0 cells as fusion partners. After primary screening and subcloning with the immunogen, two monoclonal candidates in particular, 71A1 and 1G5, showed high specificity for the cyclized peptide but not for the linear peptide. Initial characterization of these antibodies showed that they have high specificity for synthetic and endogenous multimeric forms of Aβ and do not bind to monomeric Aβ

[44] .

[0221] reagent Synthetic β-amyloid peptides 1-40 and 1-42 were purchased from Anaspec. Aβ1-40 with serine 26 substituted with cysteine ​​(Aβ S26C) was purchased from Keck Biotechnology Center (Yale University, New Haven, CT). Amyloid β-derived diffusible ligands (ADDLs) [1] and S26C dimers were prepared as previously reported

[45] .

[0222] mouse Both male and female C57BL / 6J mice were used. All procedures involving mice complied with the animal welfare guidelines of Harvard Medical School and Brigham and Women's Hospital.

[0223] Preparation of soluble brain "soaked" extracts Extraction of soluble proteins from postmortem brain tissue using the "soaking" method was performed as previously described

[11] . Briefly, cortical gray matter was dissected from freshly thawed coronal slices and then chopped into 0.5 mm wide intervals with a McIlwain tissue chopper. Chopped tissue pieces were weighed and added to extraction buffer (25 mM Tris, 150 mM NaCl, 5 μg / ml leupeptin, 5 μg / ml aprotinin, 2 μg / ml pepstatin, 120 μg / ml Pefabloc, 5 mM EDTA, 5 mM NaF, pH 7.2) at 1:5 weight:volume. Tissue pieces were soaked by swirling in 50 ml Eppendorf Protein LoBind tubes for 30 min at 4°C. Tissue debris was removed by centrifugation at 2,000g for 10 min at 4°C in a Fiberlite F14-14x50cy rotor in a Sorvall Lynx 6000 benchtop centrifuge (Thermo Fisher). Approximately the top 90% of the supernatant was removed and then centrifuged at 40,000 rpm for 110 min at 4°C in an Optima L90K ultracentrifuge (Beckman Coulter) in a SW41Ti rotor. Approximately the top 90% of the supernatant was retained as the "soaked extract" for further studies. Brain tris-buffered saline (TBS) soaked extract was aliquoted into 1.5 ml Eppendorf protein LoBind tubes and stored at -80°C.

[0224] Human biofluids and brain tissue Human CSF and plasma were obtained from two clinical cohorts: one from the BWH Division of Cognitive and Behavioral Neurology and the other from the Mayo Clinic Study of Aging. Table 1 shows the demographic characteristics and further details of both cohorts. BWH patients referred for diagnostic lumbar puncture consented to donating plasma samples and additional CSF, as well as access to their medical records under BWH IRB approval. Blood was collected into EDTA tubes, centrifuged at 1500g for 15 min, and plasma was aliquoted and frozen at -80°C, all within 3 h of collection. CSF was aspirated directly into polypropylene tubes (Sarstedt). A portion of the CSF was analyzed using the ADmark panel (Aβ 1-42 The remaining portions were immediately frozen on dry ice and then thawed and aliquoted at a later date. Clinical diagnostic information was obtained from chart review by a board-certified behavioral neurologist prior to the ADmark or study ELISA results being known.

[0225] The Mayo Clinic Study of Aging (MCSA) is a population-based prospective study of residents living in Olmsted County, Minnesota. Details of the study design and participant recruitment have been published [46, 47]. In 2004, Olmsted County residents aged 70–89 years were enumerated using the Rochester Epidemiology Project (REP) medical record linkage system as described

[48] . In 2012, the MCSA was expanded to include residents aged 50 years and older. The present analysis included 73 Mayo participants in whom we measured plasma Aβ multimers as described herein. Subject enrollment, sample collection, and sharing of samples between facilities were approved by Mayo Clinic and Olmsted Medical Center. The study protocol was approved by the Institutional Review Boards of Mayo Clinic and Olmsted Medical Center, and written informed consent was obtained from all participants.

[0226] Human brain tissue was obtained from deceased donors with probable AD undergoing diagnostic autopsy at BWH or MGH. One hemisphere was fixed for diagnosis, and the other hemisphere was sliced ​​coronally and frozen at -80°C. All human subject studies were approved in advance by the Massachusetts General Brigham Institutional Review Board, and informed consent was obtained for all human subjects.

[0227] [Table 2]

[0228] Electrophysiological extracellular field recordings Experiments were performed as previously described

[13] . Briefly, mice (1–3 months old) were deeply anesthetized with halothane and decapitated. Transverse sharp hippocampal slices (350 μm) were cut in ice-cold oxygenated sucrose-enhanced artificial cerebrospinal fluid (aCSF) containing 206 mM sucrose, 2 mM KCl, 2 mM MgSO4, 1.25 mM NaH2PO4, 1 mM CaCl2, 1 mM MgCl2, 26 mM NaHCO3, 10 mM D-glucose, pH 7.4. After dissection, slices were incubated in aCSF containing the following (in mM): 124 NaCl, 2 KCl, 2 MgSO4, 1.25 NaH2PO4, 2.5 CaCl2, 26 NaHCO3, 10 D-glucose (pH 7.4), saturated with 95% O2 and 5% CO2, and allowed to recover for at least 90 min before recording. Recordings were performed in the same solution at room temperature in a chamber submerged in aCSF. Standard procedures were used to record field EPSPs (fEPSPs) in the CA1 region of the hippocampus. Test stimuli were applied at a low frequency (0.05 Hz) of stimulation intensity that evoked fEPSP amplitudes that were 40-50% of the maximum, and test responses were recorded for 10 min before starting the experiment to ensure stability of the response. Once a stable test response was obtained, 0.5 mL of AD brain TBS-immersed extract was added to the 9.5 mL aCSF perfusate for experimental processing, and baseline was recorded for an additional 30 min. For anti-Aβ antibody experiments, 71A1 antibody was added to an aliquot of AD brain extract and incubated for 30 min with mixing, and then the mixture was added to brain slice perfusion buffer. To induce LTP, two successive 100 Hz stimuli (1 s duration) were applied to the slices with a 20 s interval. Traces were obtained with pClamp11 and analyzed using Clampfit11. Data analysis was as follows: fEPSP amplitude was measured using the initial fEPSP slope, and three successive slopes (1 min duration) were averaged and normalized to the mean value recorded 10 min before pretreatment stimulation. Data are presented as mean ± SEM. Significant differences were determined using one-way ANOVA test with post hoc Tukey's test.

[0229] Electrophoresis and WB Samples were loaded onto 4-12% or 12% Bis-Tris gels (SurePAGE, Genscript) using MES-SDS running buffer (Invitrogen), transferred to nitrocellulose membranes, and probed for various proteins using standard WB. The resulting blots were detected by ECL and signals were captured on film.

[0230] Immunoprecipitation 800 μL of human brain TBS-soaked extract or CSF sample was incubated with 10 μg of antibody for 1 h at 4 °C. Immunoprecipitates (IPs) were incubated with protein G magnetic beads (Bio-Rad) overnight at 4 °C and then washed three times with TBS. Post-IP solutions were also saved for analysis. Immunoprecipitated proteins were then eluted with 8 M guanidine hydrochloride (GnCL) (Thermo-Fisher).

[0231] Size Exclusion Chromatography (SEC) Brain TBS-soaked extracts or CSF (total volume 350 μL) were injected onto a Superdex200 Increase and run on a fast protein liquid chromatography (FPLC) instrument (AKTA; GE Healthcare) in TBS, pH 7.4. Fractions of 500 μL were collected for downstream experiments. The column was calibrated with gel filtration standards (Bio-Rad) ranging from 1,350 to 670,000 Da.

[0232] Affinity purification using 71A1 Brain TBS soaked extracts (2 mL) were mixed with high capacity streptavidin agarose resin (Pierce) pre-conjugated with 50 ug biotinylated 71A1 for 2 h at room temperature (RT) with swirling. Beads were extensively washed with 10 column volumes of SMCxPRO wash buffer and eluted with 0.2 M glycine (pH 3) followed by neutralization with 1 M Tris-HCl (pH 8.5). Affinity purified material was desalted with PBS (pH 7.4) using a PD MidiTrap G-25 before application to hippocampal slices.

[0233] MSD ELISA Human brain extracts, CSF, immunoprecipitated samples, and SEC fractions were each diluted with 1% BSA in wash buffer (TBS supplemented with 0.05% Tween). For our home-made Meso Scale Discovery assay (MSD) electrochemiluminescence platform, each well of an uncoated 96-well multi-array plate (Meso Scale Discovery, #L15XA-3) was coated with 30 μL of PBS solution containing capture antibody (for all human Aβ ELISAs, 3 μg / mL 266, a monoclonal antibody that recognizes the middle region of Aβ [mouse analog of solanezumab]), incubated overnight at room temperature, followed by blocking with 5% BSA in wash buffer for 1 h at room temperature with shaking at >300 rpm. The detection antibody solution was prepared with biotinylated detection antibody, 100 ng / mL streptavidin sulfo-TAG (Meso Scale Discovery, #R32AD-5), and 1% BSA diluted in wash buffer. Following the blocking step, 50 μL / well of sample followed by 25 μL / well of detection antibody solution were incubated for 2 h at room temperature with shaking at >300 rpm, washing wells with 150 μL of wash buffer between incubations. Plates were read and analyzed according to the manufacturer's protocol. Antibodies used for detection of specific antigens were 139-5 (rabbit recombinant, Biolegend) for hAβ (1-40 specific) and D3E10 (rabbit recombinant, Cell Signaling Technology) for hAβ (1-42 specific).

[0234] SMCxPRO Immunoassays The SMCxPRO platform (Sigma Millipore) is based on single molecule counting technology and typically allows for a 20- to 100-fold increase in sensitivity compared to conventional detection systems. Using a kit from Sigma Millipore, biotinylated capture mAbs (1C22, 1G5, and 71A1) were conjugated to streptavidin magnetic particles (MPs) (Dynabeads MyOne, Thermo Fisher Scientific) at a ratio of 12.5 μg of biotinylated antibody per milligram of MPs. Capture mAb-bound MPs were diluted to 50 μg / mL in Aβ multimer assay buffer (Tris buffer; 50 mM Tris, 150 mM NaCl, pH 7.6) with 1% Triton X-100, 0.0005% (weight / volume) d-des-thio-biotin, and 0.1% bovine serum albumin). 50 μL of this suspension was added to 150 μL of sample, standard, or blank and incubated for 2 h at 600 rpm in a shaking incubator at 25° C. The MPs were isolated using a magnet and unbound material was removed by washing with 1× SMC wash buffer using a HydroFlex plate washer (Tecan Group AG, Männedorf, Switzerland). Fluorescently labeled (Alexa-647 dye) detection antibody 3D6 (20 μL, 200 ng / mL) was added to each well. The MPs with antibody-multimeric Aβ sandwich were then incubated for 1 h at 25° C. with agitation using a Jitterbug shaker (Boekel, Feasterville, PA, USA). Unbound detection reagent was removed by washing four times with wash buffer. The wash buffer was removed by aspiration and the fluorescently labeled 3D6 detection antibody was released by shaking with elution buffer B (11.5 μL / well) for 10 min at 25° C. 11 μL of the eluate was then transferred to wells of a clean 96-well plate containing Neutralization Buffer D (11 μL / well). The neutralized samples (20 μL / well) were then transferred to a black 384-well reading plate (Aurora) and read on an SMCxPRO instrument. When the fluorescently labeled antibodies are excited with a 642 nm laser and passed through the interrogation space, they emit light that is measured using a confocal microscope lens and a photon detector.The output from the detector is a train of pulses, with each pulse representing one detected photon. The lower limit of confidence quantification (LLoQ) was defined to be the smallest inversely interpolated standard that provided a signal twice the background, with a calculated recovery percentage between 80% and 100% and a coefficient of variance (CV) ≤ 20%.

[0235] immunohistochemistry Fresh or thawed (from frozen stocks) human brain blocks were embedded in Tissue-Tek® OCT. The compound was frozen overnight at -80°C. Before sectioning at 20-30 μm thickness using a cryostat (Leica), the frozen blocks were changed to -20°C for 2 h to soften the tissue for sectioning. Frozen sections were then directly mounted on MAS-GP™ Adhesion microscope slides (Matsunami) and stored at 4°C until staining. For 3,30-diaminobenzidine (DAB) staining, frozen sections were equilibrated in PBS containing 0.3% Triton-X100 (PBST) for 30 min, followed by blocking of endogenous peroxidase activity and antibody nonspecific binding for 1 h each. Primary antibodies were diluted in PBST and incubated with the sections overnight at 4°C. After three washes with PBST, the sections were incubated with biotinylated secondary antibodies for 1 h. Immunoreaction products were visualized by incubation with 1) DAB containing nickel ammonium sulfate as an enhancing agent or 2) Vina Green chrome (Biocare Medical). Stained sections were viewed using an Axioskop2 (Zeiss).

[0236] Quantification and statistical analysis All statistical analyses were performed using GraphPad Prism 9 software. Statistical details of the experiments are described in the text and / or figure legends.

[0237] [Example 4.1] Immunogen design and monoclonal antibody generation of 71A1 and 1G5 The monoclonal antibodies 71A1 (a subclone of the parent clone 7A1a) and 1G5 were raised against synthetic conformational peptide immunogens designed to mimic the potential three-dimensional structure formed upon dimerization of monomeric amyloid β protein (Aβ). 1-40 A peptide immunogen comprising amino acid residues 9-18 of Aβ was synthesized, cyclized, and post-synthetically modified to enable folding into a stable dimer-like entity (patent pending). The rationale behind selecting residues 9-18 of the Aβ peptide was that the amino acids in this region were thought to be more likely to reassociate into a unique three-dimensional structure that is present only in the aggregated form of Aβ.

[0238] Balb / c mice were immunized with this cyclized conformational peptide prior to splenectomy using a maleimide-activated keyhole limpet hemocyanin (KLH) carrier complex for primary and secondary immunizations, and a proprietary helper 2 T cell epitope for subsequent boosts. Fusions were performed using immunized mouse splenocytes and mouse myeloma F0 cells as fusion partners. After primary screening with immunogens and subcloning, two monoclonal antibody candidates in particular (71A1 and 1G5) showed high specificity for the cyclized peptide but not for the linear peptide. Initial characterization of these antibodies showed high specificity for synthetic and endogenous multimeric forms of Aβ and no binding to monomeric Aβ

[44] .

[0239] [Example 4.2] 71A1 and 1G5 recognize Aβ biochemically and histologically in human brain We tested two of these novel antibodies, 1G5 and 71A1, against natural sources of human Aβ. First, we obtained highly diffusible Aβ species without homogenization from minced brain pieces of neuropathologically typical AD cortex incubated in Tris-buffered saline (TBS) for only 30 min using a recently developed method called "soaking extraction"

[11] . These diffusible aqueous extracts have been shown to retain most of the synaptotoxic activity of AD cortical samples. Subsequent homogenization of the soaked brain pieces yields more Aβ, which has little synaptotoxic activity

[11] . The soaking procedure is illustrated in Figure 3A. We performed immunoaffinity pull-down from AD brain soaked extracts using 1G5 and 71A1 together with the positive control antibodies 4G8 (mainly anti-monomer) and 1C22 (anti-multimer) and the negative control normal mouse IgG. Both 1G5 and 71A1 pulled down much less Aβ from AD brain soaked extracts compared to 4G8 and 1C22, as judged by immunoblots probed with two rabbit monoclonal recombinant antibodies (mAbs) raised against different Aβ epitopes, but consistently pulled down more than mouse IgG, which did not pull down Aβ (Figure 3B). As soluble oAβ disassembles into monomers and to some extent covalently linked dimers on denaturing SDS-PAGE

[49] , we observed immunoblot signals around and below 6-7 kDa (Figure 3B, lower panel (longer exposure)). To confirm this finding, we then used our highly specific in-house Aβ x-40 and x-42 monomer immunoassays [50, 51] to quantify Aβ content pulled down from three individual AD brains by 1G5, 71A1, 1C22 (positive control), and mouse IgG (negative control).As shown in Figure 3C (Aβ pulled down with protein G beads was then denatured with guanidine hydrochloride (GnCl)) and Figure 3D (Aβ remaining in the post-IP supernatant was then denatured with GnCl), both 1G5 and 71A1 pulled down small but consistent amounts of Aβ from AD brain soaked extracts, always more than the negative control mouse IgG but much less than 1C22. This result was consistent with the immunoblot data (Figure 3B). To also show that the pulldown and immunodepletion by the four antibodies was consistent, we calculated the Aβ mass (in ng, measured against ADDL standards) from three AD brains obtained at and after IP with the four antibodies.

[0240] Having thus established that 1G5 and 71A1 could bind Aβ from highly soluble human brain extracts, we used immunohistochemistry to examine whether 1G5 and 71A1 could label Aβ plaques in human brain. When we first tested 1C22 for use in immunohistochemistry, we found that 1C22 was unable to stain typical PFA-fixed AD brain sections, but could readily stain unfixed frozen sections (Figure 4A). Furthermore, treatment of the latter sections with 4% PFA for 20 min reduced most of the 1C22 immunoreactivity (Figure 4A). This phenomenon is common for antibodies that recognize conformational epitopes. For 1G5 and 71A1, we found that 1) both antibodies labeled extracellular amyloid plaques (i.e., deposited Aβ) in unfixed frozen sections in a similar pattern, and 2) 4% PFA treatment for 20 min substantially reduced the labeling (Figure 4A).

[0241] To further establish that these antibodies labeled parenchymal Aβ, we used double immunostaining. Frozen sections were sequentially labeled with D54D2 (for the Aβ N-terminal region, representing total Aβ visualized by DAB) and then with either 1C22 or 71A1 (visualized by Vina Green). In Figure 4B, the left panel shows that only D54D2 labeled amyloid plaques, the middle panel shows amyloid plaques double-labeled with D54D2 and 1C22 or 71A1 (vina Green) for total Aβ (DAB), and the right panel shows double-labeled plaques at higher magnification. These findings support that 71A1 and 1G5 recognize Aβ brain deposits in situ. We also tested 1C22, 1G5, and 71A1 on plaque-free control brains to show specificity for Aβ. All three antibodies were unable to detect any signal (data not shown).

[0242] Because the immunoreactivity of 1G5 and 71A1 was similar to the previously shown multimer-preferring 1C22 [52, 53], we next investigated whether the two new antibodies could protect against Aβ-induced synaptic toxicity of soluble AD brain extracts. Using electrophysiology of wild-type (wt) mouse brain hippocampal slices

[54] , we found that 71A1 added to the slice perfusate at 2.12 μg / mL completely prevented the inhibition of hippocampal LTP by AD-soaked extracts, but had no effect on LTP by itself (Figure 4C). Specifically, fEPSP slopes in artificial cerebrospinal fluid (aCSF) vehicle alone were 152.6 ± 5.3% (N = 6); in aCSF + AD extract: 115.7 ± 4.9% (N = 5); in aCSF + 71A1 alone: ​​149 ± 8.3% (N = 4); and in aCSF + AD extract premixed with 71A1: 154.9 ± 9.8% (N = 4). This effect mimicked the oAβ neutralization benefits of mAbs 3D6 and 82E1 that we previously reported

[13] . Furthermore, we used 71A1 to affinity purify oAβ from the same AD brain soaked extracts and tested the synaptic toxicity of purified oAβ on wt mouse brain hippocampal slices. Affinity purified oAβ also significantly inhibited hippocampal LTP (Figure 4D). 1) statistical analysis of the difference between AD brain extract alone and AD brain extract premixed with 71A1 confirmed that 71A1 significantly rescued the LTP deficit caused by the extract (Figure 4E), and 2) statistical analysis of the difference between 71A1-purified oAβ and aCSF control showed that 71A1-purified oAβ significantly inhibited LTP, similar to the input-soaked extract (Figure 4F). Collectively, these data indicate that 71A1-reactive Aβ species confer synaptic toxicity that can be counteracted by 71A1, despite oAβ being a minority population in AD brain as shown by immunoprecipitation with protein G (Figures 3B-3D).

[0243] [Example 4.3] 71A1 and 1G5, inversely to 1C22, preferentially bind Aβ species in CSF compared to brain Next, we investigated the extent to which 1G5 and 71A1 could recognize Aβ from another natural source, human CSF. Using a similar experimental setup as used for affinity pull-down from brain-soaked extracts (Figures 3C, 3D), we used 1G5, 71A1, 1C22, and mouse normal IgG (control) to perform pull-down from three individual CSF samples derived from AD patients collected at the Memory Disorders Clinic at Brigham and Women's Hospital. 1G5 and 71A1 each immunoprecipitated surprisingly high amounts of Aβ, as measured by Aβx-40 and x-42 monomer immunoassays after GnCL denaturation (Figure 5A, top panel), with correspondingly reduced amounts of Aβ in the post-IP supernatant (Figure 5A, bottom panel). Moreover, in contrast to the soluble AD brain extracts studied above, both 1G5 and 71A1 pulled down higher relative amounts of Aβx-42 from CSF than 1C22. This suggests that there may be differences in the native Aβ population between human brain and CSF. We also found that 71A1 showed higher Aβ pull-down efficiency than 1G5 in all three CSF samples (Figure 5A). Therefore, we decided to use 71A1 to perform IP-ELISA on 19 different CSFs from the same BWH clinic (see Table 1 for patient demographics and diagnoses). 71A1 immunoprecipitated Aβ from all of the CSF samples (Figure 5B). We next investigated whether this IP-ELISA data correlated with commercially available ADmark Aβ1-42 levels obtained from the same CSFs by our clinic. The Aβx-42 signal from our 71A1 immunoprecipitates (IPs) (after GnCl denaturation) correlated significantly (R 2= 0.43, p = 0.0046) (Figure 5C), confirming the specificity and reliability of our 71A1 affinity purification. Here, we also calculated the Aβ mass (ng) from the three CSFs in Figure 5A, IP and post-IP with the four antibodies. Unlike the case of brain soaked extracts, we found that the IP+post-IP of the four different antibodies shared the same trend across the three individual CSF samples, but were not completely consistent. This finding prevented us from further using IP-ELISA to accurately quantify oAβ from biofluid samples.

[0244] The above results highlight the technical limitations of these Aβ IP-ELISA approaches for quantifying the relative levels of 71A1-reactive Aβ species in numerous brain, CSF, and potentially plasma samples. Therefore, we proceeded to develop a sandwich immunoassay to more accurately quantify apparent Aβ multimers in biofluids. In this context, we previously quantified 1C22-reactive Aβ multimers in human CSF, but 1C22 pulled down small amounts of CSF Aβ multimers (Figure 5A), even with the highly sensitive Erenna immunoassay system (Millipore), forcing us to use undiluted (neat) CSF to obtain sufficient levels from all samples (Figure 5A). Taking advantage of the unique properties of 71A1 (described above), which has a higher binding capacity for Aβ multimers in CSF than in brain extracts (in stark contrast to 1C22), we designed a sandwich immunoassay using 71A1 as a capturer to better quantify oAβ levels in biological fluids such as CSF and ultimately plasma.

[0245] [Example 4.4] The 71A1 / 3D6 assay provides highly sensitive and specific quantification of Aβ multimers We developed a quantitative sensitive immunoassay for the detection of 1G5- and 71A1-reactive Aβ species in human CSF and plasma. Similar to our 1C22 / 3D6 oAβ sandwich ELISA established on the Erenna (Millipore) platform [30, 31], we used biotinylated 1G5 or 71A1 as capture antibodies conjugated to magnetic streptavidin-coated beads, and 3D6 (labeled with Alexa-647 dye) as detector antibody on the bead-based immunoassay platform SMCxPRO (an upgraded version of Erenna from Millipore) (Figure 6A). As shown in Figures 6B-6D, when we used conventional calibrators of synthetic ADDLs [1], both the 71A1 / 3D6 and 1G5 / 3D6 assays showed 1) an LLoQ of 0.6 pg / mL, the same as our previously developed 1C22 / 3D6 multimer assay

[30] ; 2) recoveries calculated to be 80-120%; and 3) intra-assay CVs well below 20% across the ADDL concentration range of 0.6-80 pg / mL, which easily covers the entire range of detectable analyte concentrations at the appropriate dilutions used in CSF and plasma (see below). As observed in our IP-ELISA assay, where 71A1 outperformed 1G5 in terms of pull-down efficacy and consistency (Figure 5A), we focused on validating the specificity of the 71A1 / 3D6 assay for multimeric Aβ found in human biofluids.

[0246] To assess whether 71A1 preferentially recognizes oAβ over monomeric Aβ, we used a synthetic S26C Aβ40 dimer covalently linked via a disulfide bond

[45] . This dimer can be converted back to a monomeric form by reducing agents such as DTT. Before comparing the new 71A1 / 3D6 assay and the previous 1C22 / 3D6 assay for this Aβ40 dimer, we first showed that the two assays have the same sensitivity to ADDLs (Figure 6E). Both the 1C22 / 3D6 oAβ assay and the 71A1 / 3D6 assay showed a strong and significant binding preference for the non-reduced S26C Aβ40 dimer (confirmed by SDS-PAGE in Figure 6F) over their reduced monomeric counterparts (Figures 6G-6H). This result suggests that both 1C22 and 71A1 antibodies bind preferentially to conformational Aβ, while binding to monomeric Aβ at 100-fold less. Furthermore, the 71A1 / 3D6 assay showed much higher sensitivity to S26C Aβ40 dimers than the 1C22 / 3D6 assay. 8 pg / mL of S26C Aβ40 dimers in the 71A1 / 3D6 assay showed a similar signal to 1.5 ng / mL of S26C Aβ40 dimers in the 1C22 / 3D6 assay (Figure 6G vs. 6H; note the different units on the horizontal axis). This may help explain the observed difference between 71A1 and 1C22 in terms of their respective binding preferences for AD brain-derived oAβ vs. oAβ in CSF (compare Figures 3C and 5A).

[0247] Having assessed the sensitivity and specificity of the 71A1 / 3D6 assay for synthetic multimeric versus monomeric Aβ, we proceeded to test whether the 71A1 / 3D6 assay could easily and accurately measure oAβ from natural sources such as human brain soaked extracts. We serially diluted human brain soaked extracts (tissue:soaking buffer=1:5 wt / vol) 1000-fold, 2000-fold, 4000-fold, and 20,000-fold, and measured the diluted samples with the 71A1 / 3D6 assay (Figure 6I). We found that the signal from the diluted brain soaked extract samples was fully dilutable, with an average recovery % of 93.3% at 20,000-fold (Figure 6I, bottom panel). This dilution linearity test of the 71A1 / 3D6 assay demonstrated its ability to measure oAβ from natural biological material over a wide concentration range.

[0248] [Example 4.5] The 71A1 / 3D6 assay recognizes high molecular weight Aβ multimers in the brain Next, we investigated which types of native oAβ species are detected by the 71A1 / 3D6 immunoassay. We used non-denaturing size-exclusion chromatography (SEC) to fractionate human AD brain immersion extracts on a Superdex 200 Increase high-resolution column calibrated with molecular weight (MW) standards (Figure 7A). We performed the 71A1 / 3D6 assay on 30 SEC fractions starting from the void volume of the column (fractions of the UV280 chromatogram in Figure 7B). The 71A1 / 3D6 signal (measured with ADDL calibrators in pg / mL) peaked at fraction numbers 3-6 within the void volume range. This indicates that the estimated analyte size is at least 670 kDa (Figure 7C). To confirm that this 71A1 / 3D6 signal in fractions 3-6 actually represents Aβ, we denatured 30 fractions using 8 M GnCl and then measured them with a well-established monomeric Aβ42 immunoassay (capture: m266, detector: D3E10, MSD platform) [11, 50]. We found that GnCl-released monomeric Aβ species peaked in fractions 3-6, the same as the 71A1 / 3D6 assay (compare Figure 7C with Figure 7D [GnCl(+) = light gray bars, right side of each pair of bars]). We found that the native, monomeric species (Figure 7D: GnCl(-) = dark gray bars, left side of each pair) eluted primarily in fractions 19-26 by the same Aβ monomer assay. Taken together, these data suggest that the abundant 71A1 / 3D6 signal observed in fractions no. 3–6 of the AD brain soaked extract represents higher order, high MW soluble Aβ multimers that can be denatured to Aβ monomers by GnCl, and that this assay shows very poor detection of monomers, similar to the pattern previously detected in the 1C22 / 3D6 oAβ assay [ 30 , 53 ].

[0249] [Example 4.5] The 71A1 / 3D6 assay quantifies low molecular weight Aβ multimers in human CSF To validate the 71A1 / 3D6 assay for human CSF, we performed a dilution linearity study similar to the steps used with brain soaked extracts (Figure 8A). We observed a mean recovery of 98.94% at 1:2 and 1:4 dilutions, a range in which the measurements were accurate and stable, but at 1:8 dilution (Figure 8A), the dilution recovery was suboptimal. The reason for this is unclear. We proceeded to use the 71A1 / 3D6 assay to measure CSF derived from a cohort of 36 subjects with various neurological diagnoses (Table 1: Patient demographics and diagnostic information) recruited at the Memory Disorders Clinic (Brigham and Women's Hospital). All 36 samples were quantified by the 71A1 / 3D6 assay with a mean level of 5.47 ng / mL, ranging from 1.75 to 20.38 ng / mL (Figures 8B-8D). We investigated whether these CSF 71A1 / 3D6 levels correlated with other biomarkers of AD pathology in CSF, as measured by the widely used, commercially available ADmark clinical assay (Athena Diagnostics). ADmark data were available for 29 of the 36 samples, so correlations were performed on these 29 samples. There was no statistically significant correlation between the 71A1 / 3D6 oAβ signal and ADmark Aβ1-42 monomer levels in these CSF (Figure 8B), but there was a significant correlation between the 71A1 / 3D6 signal and ADmark total tau (R 2 = 0.41, p = 0.0002 and phospho-tau (pT181) (R 2= 0.42, p = 0.0001; Pearson correlation coefficient) (Figures 8C-8D). Similar to our analysis of brain soaked extract SEC fractions (Figures 7A-7D), we performed the 71A1 / 3D6 assay on 30 SEC fractions of human CSF starting from the void volume of the column (UV280 chromatograms are shown in Figure 8E). The 71A1 / 3D6 signal (measured with ADDL calibrator in pg / mL) showed two peaks: a smaller peak numbered 3-6 and a larger peak numbered 24-28 (Figure 8F). This was a different distribution than the brain soaked extract (Figure 7C). Taken together, these data indicate that the 71A1 / 3D6 assay can quantify low molecular weight Aβ multimers found in human CSF in addition to the high molecular weight multimers found in brain soaked extracts.

[0250] [Example 4.6] The 71A1 / 3D6 assay quantifies Aβ multimers in human plasma CSF is not at all attractive for biomarker development because its collection is considered too invasive for many people and it is relatively complicated and expensive to handle. Moreover, serial lumbar puncture measurements are very rarely performed. In contrast, blood sampling is routinely performed, minimally invasive, and inexpensive. Therefore, we sought to establish the accuracy of the 71A1 / 3D6 assay in plasma, a highly complex matrix that poses technical challenges to accurate quantification by immunoassay. We tested the degree of matrix interference in plasma by 1) plasma dilution and recovery, 2) analyte spike recovery, and 3) immunodepletion. Compared to 4-fold dilution, the 71A1 / 3D6 assay showed nearly 100% dilution recovery (average 96.6%) at up to 16-fold dilution of each of six individual plasmas (from the Mayo Clinic Alzheimer's Disease Research Center) (Figure 9A). We also observed a matrix effect of plasma only when using neat plasma (no dilution) in this assay (Figures 9F-9G). Therefore, we chose to use a 1:8 dilution in the following experiments. We performed analyte spike recovery experiments on 8-fold diluted plasma samples using three different sources of native oAβ from AD subjects: brain homogenate, brain soaked extract, and CSF. This resulted in excellent average recovery values ​​of 97.5%, 92.9%, and 99.7%, respectively (Figure 9B). We then attempted immunodepletion experiments on individual 8-fold diluted plasmas using biotinylated antibodies and streptavidin-coated plates. 71A1 immunodepleted an average of 61.5% of the 71A1 / 3D6 signal, whereas 1C22 immunodepleted only 15.6% of the 71A1 / 3D6 signal (Figure 9C). This indicates that the 71A1 / 3D6 assay is specific for detecting oAβ in plasma. Furthermore, we investigated the specificity of the 71A1 / 3D6 assay for Aβ multimers versus monomers in brain homogenates, CSF, and plasma.We measured these samples with or without multimer denaturing treatment with 8 M GnCl and found that GnCl reduced the 71A1 / 3D6 signal from brain homogenate by >70%, from plasma by >84%, and abolished the signal from CSF (>99%) (Figure 9D). To exclude any negative effect of GnCl on the immunoassay, all samples were heavily diluted before the assay (final concentration of GnCl was <0.25 M).

[0251] In summary, all data from plasma dilution and spike-recovery experiments with three different natural oAβ sources derived from AD subjects showed no significant matrix interference in the plasma 71A1 / 3D6 assay. Furthermore, immunodepletion experiments highlight the ability of 71A1 to bind to its target in individual human plasma. Therefore, we performed the 71A1 / 3D6 immunoassay on 8-fold diluted plasma samples from a cohort of 73 cognitively normal individuals (Mayo Clinic Alzheimer's Disease Research Center). The mean dilution-adjusted concentration based on the synthetic oAβ (ADDL) standard curve was 43.34 ± 29.09 pg / mL (Figure 9E, left). The CVs of these quadruplicate plasma samples were ≦20% except for five subjects (Figure 9E, right), indicating the consistency of the plasma assay.

[0252] [Example 5] Polymer-directed Aβ antibodies reduce synaptic toxic polymeric Aβ in the brain and improve behavioral abnormalities in humanized APP knock-in mice Strong evidence exists that soluble multimers of amyloid beta protein (oAβ) help initiate the pathogenic cascade in Alzheimer's disease (AD), suggesting therapeutic strategies that target oAβ rather than monomeric or fibrillar Aβ. The new antibody 71A1 is approximately 100-fold more sensitive to oAβ than synthetic monomers. Substances that 71A1 specifically immunoprecipitates from AD soluble brain extracts impair synaptic function similarly to full extracts. Consistent with that, preincubation of brain extracts with 71A1 neutralizes its synaptic toxicity. 71A1 has potential intrinsic activity against disease-associated oAβ, making it a novel candidate for AD treatment.

[0253] method 15 mg / kg of 71A1 or anti-KLH (negative control) was administered ip weekly to 45 humanized APP knock-in mice (APP NLGF / NLGF) from 8 to 20 weeks of age. Cognition was assessed by spontaneous alternation (Y-maze). Brains were harvested at 21 weeks of age for biochemical, electrophysiological, and immunohistochemical analyses. Diffusible ("soak") extracts of brain were prepared to measure monomeric and polymeric Aβ using a homemade ultrasensitive assay. Aliquots of the same soak extracts were used to treat wild-type mouse hippocampal slices to measure long-term potentiation (LTP) to assess APP after treatment. NLGF / NLGF Synaptic toxicity in the brain was assessed.

[0254] result 6 month old APP NLGF / NLGFoAβ derived from APP NLGF male mouse brains impaired LTP in wild-type mice, which was rescued by treatment with 71A1 (see Figures 10A-10C). As shown in Figure 11A, 71A1 treatment significantly improved Y-maze performance in male mice, but not in female mice, compared to control antibody treatment. At that time, 71A1 treatment reduced oAβ load in male mouse brains (trend without statistical significance, Figure 11B). In addition, 71A1 treatment reduced synaptic toxicity in APP NLGF male mouse brains, as reflected by failure to inhibit LTP in wild-type mouse hippocampus (see Figures 12A-12B).

[0255] These results indicate that 71A1 is a multimer-preferential monoclonal antibody as a potential therapeutic for AD, reducing brain oAβ and oAβ-induced synaptic toxicity. We believe this provides the first proof-of-concept mouse study using a carefully characterized antibody with oAβ preference. We observed that 1) there was a correlation between oAβ concentration and its synaptic toxicity, and 2) 71A1 improved aspects of cognitive impairment in APPNLGF / NLGF mice by targeting oAβ.

[0256] [Example 6] Identification of peptidomimetics that bind to the 71A1 variable region The New England Biolabs (NEB) Ph.D.-12™ Phage Display Peptide Library Kit (with accompanying instructions) was used to isolate amino acid residue peptide ligands (herein referred to as peptidomimetics) that bound to immobilized Mab 71A1. The New England Biolabs Ph.D.-12™ Phage Display Peptide Library Kit is a combinatorial library of random 12 amino acid peptides (12-mers) fused to the minor coat protein (pIII) of M13 bacteriophage. The peptides were expressed as fusions with the coat protein resulting in the display of the fusion protein on the surface of the bacteriophage. The library consisted of 2.7×109 electroporated sequences and was amplified once to obtain approximately 55 copies of each sequence in 10 μl of feed phage.

[0257] Immobilized 71A1 antibody was used to isolate the 12 amino acid peptide shown in FIG. 13 that bound to the variable region. Thermo Scientific™ AminoLink Plus coupling resin was used to immobilize Mab 71A1. Negative control particles were made using monoclonal antibody 3C7 with specificity for an unrelated peptide sequence. The Thermo Scientific™ AminoLink Plus coupling resin protocol used to generate 71A1 particles panned with the NEB library is provided. Briefly, 2 mL of aminolink particle slurry was washed twice with 10 mL of 0.1 M sodium bicarbonate buffer pH 10 by centrifugation at 2000×g for 10 min in a 15 mL conical tube. 10 mg of 71A1 antibody in this binding buffer was then added to the particles transferred to a 2 mL eppendorph tube and rotated for 2 h at room temperature. The slurry was then added to a Millipore Ultrafree-MC centrifugal filter unit and the resin was washed three times with 2 mL of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 binding buffer. The centrifuged particles were then mixed with 2 mL of pH 7.2 buffer and 40 μl of 50 mM sodium cyanoborohydride solution and rotated for 4 hours. The aminoLink gel contains aldehyde functional groups that react spontaneously with the primary amines of the antibody. The formed Schiff base bonds are then reduced to stable secondary amine bonds in the presence of sodium cyanoborohydride, a mild reducing agent. The particles were then washed twice with 2 mL of 1 M Tris HCL, pH 7.4, resuspended in 2 mL of this same buffer and 40 μl of sodium cyanoborohydride to block unbound sites on the particles and rotated for 30 minutes. The particles, washed five times with 1 M sodium chloride, were suspended in 1 mL of PBS.

[0258] First, the library (10 μl) was mixed with isotype-matched, variable region-irrelevant immobilized 3C7 antibody particles (200 μl added to a Millipore Ultrafree-MC centrifugal filter unit) to remove nonspecifically bound bacteriophage. The library was removed from these control particles by centrifugation at 2000×g and then mixed with immobilized 71A1 antibody (200 μl) in a new Millipore Ultrafree-MC centrifugal filter unit, after which unbound phages were washed away by centrifugation at 2000×g. The particles were then washed with 1 mL of PBS, and this procedure was repeated several times. Specifically bound phages were eluted from the immobilized antibody using 100 μl of 0.1 M glycine-HCl, pH 2.0. Bacteriophage were amplified by infection and growth of liquid cultured E. coli bacteria as described in the protocol provided with the kit and included herein. Isolated and amplified bacteriophage were then isolated from the bacterial supernatant using PEG / NaCl (20% (wt / vol) polyethylene glycol-8000, 2.5 M NaCl) precipitation. The isolated phages were amplified and the pool of phages was enriched for binding sequences against the antibody variable regions by additional binding / amplification cycles. After 3-4 rounds, individual clones were characterized by DNA sequencing (AIBioTech-American International Biotechnology Services, Richmond, VA) and subsequent amino acid predictions made from the isolated DNA sequences (Figure 13).

[0259] A standard indirect enzyme-linked immunosorbent assay (ELISA) with purified monoclonal antibody 71A1 was used to assay binding to peptidomimetics 1-13 (sequence in Figure 13) conjugated to maleimide-activated microtiter plates at 10 μg / mL in 0.1 M sodium bicarbonate buffer pH 10 and blocked with PBS and 2% BSA. After 1 hour incubation, wells were washed and then 100 ul of horseradish peroxidase (HRPO)-conjugated goat anti-mouse secondary antibody was added and plates were incubated for an additional hour before the addition of TMB (3,3',5,5'-tetramethylbenzidine) substrate. Absorbance was measured at 650 nm in a spectrophotometer. The results shown in Figures 14A-14B indicate that most, if not all, of the peptidomimetics isolated by phage display bind to monoclonal antibody 71A1. In this assay, peptides 1, 3, 5, 7, 8, 11, and 13 showed the most binding, with peptides 5, 8, and 7 being the best. References

[0260] [Table 3-1]

[0261] [Table 3-2]

[0262] [Table 3-3]

[0263] [Table 3-4]

[0264] [Table 3-5]

[0265] [Table 3-6]

[0266] [Table 3-7]

[0267] [Table 3-8]

[0268] [Table 3-9]

[0269] [Table 3-10]

[0270] [Table 3-11]

[0271] [Table 3-12]

[0272] [Table 3-13]

[0273] [Table 3-14]

[0274] [Table 3-15]

[0275] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment that specifically binds to amyloid beta protein multimers (oAβ), the antibody or antigen-binding fragment comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 sequences derived from a VH domain sequence selected from the group consisting of SEQ ID NOs: 20-22, and CDR-L1, CDR-L2, and CDR-L3 sequences derived from a VL domain sequence selected from the group consisting of SEQ ID NOs: 25-27; or (b) the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 sequences derived from a VH domain sequence selected from the group consisting of SEQ ID NOs: 30-32, and CDR-L1, CDR-L2, and CDR-L3 sequences derived from a VL domain sequence selected from the group consisting of SEQ ID NOs: 35-37; or (c) the VH domain comprises the sequence of SEQ ID NO: 19, and the VL domain comprises the sequence of SEQ ID NO: 24; or (d) the VH domain comprises the sequence of SEQ ID NO: 29, and the VL domain comprises the sequence of SEQ ID NO: 34; The antibody or antigen-binding fragment.

2. 1. A method for detecting oAβ in a sample comprising one or more biological fluids, preferably in a sample derived from a human subject, comprising: contacting the sample with a surface-bound capture antibody, wherein the capture antibody comprises the antibody or antigen-binding fragment of claim 1; contacting the sample with a labeled detection antibody comprising a detectable label, wherein the detection antibody binds to the capture antibody-αAβ complex under conditions sufficient for the detection antibody to bind to the capture antibody-αAβ complex; and detecting and optionally quantifying said detectable label, wherein the amount of said detectable label is proportional to the amount of oAβ in said sample, thereby detecting oAβ in said sample. A method comprising:

3. 3. The method of claim 2, wherein the one or more biological fluids comprise blood or plasma, and / or the surface is the surface of a plate or bead, and optionally the plate is a multi-well plate or test strip, or the beads are selected from magnetic beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, e.g., SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, or TEFLON® beads.

4. 3. The method of claim 2, wherein the capture antibody is conjugated to a biotin moiety, the surface is coated with streptavidin, and the antibody is attached to the surface via a biotin-streptavidin bond, and / or the detection antibody binds to Aβ at a different epitope than the capture antibody, and / or the capture antibody comprises an affinity tag, and the detection antibody binds to the affinity tag, and / or the detectable label is a radioisotope, a chemiluminescent substance, a fluorescent substance, a metal complex, a bioluminescent substance such as luciferase, or a nucleic acid.

5. The detection and optional quantification of the oAβ concentration in the sample can be performed using radioimmunoassay (RIA) if the detection antibody is labeled with a radioisotope, chemiluminescence immunoassay (CIA) if the detection antibody is labeled with a chemiluminescent substance, fluorescence immunoassay (FIA) if the detection antibody is labeled with a fluorescent substance, electrochemiluminescence immunoassay (ECLIA) if the detection antibody is labeled with a metal complex, or bioluminescence immunoassay (BCIA) if the detection antibody is labeled with a bioluminescent substance such as luciferase. The method according to claim 2, further comprising using a bioluminescence immunoassay (BLIA) in the latter case; using an immuno-PCR method comprising amplifying the nucleic acid labeled with the antibody by PCR and detecting the nucleic acid when the detection antibody is labeled with a nucleic acid; using a turbidimetric immunoassay (TAI) in which turbidity caused by the formation of an immune complex is detected; using a latex agglutination turbidimetric assay (LA) in which latex agglutinated by the formation of a complex is detected; or using an immunochromatography assay using a reaction on a cellulose membrane.

6. a surface, optionally a plate or bead; A capture antibody comprising the antibody or antigen-binding fragment of claim 1, optionally linked to the surface; detection antibodies, and Standards containing known concentrations of oAβ Kit including:

7. the surface is the surface of a plate or bead, and optionally the plate is a multi-well plate or a test strip; or 7. The kit of claim 6, wherein the beads are selected from magnetic beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, such as SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, or TEFLON® beads.

8. 7. The kit of claim 6, wherein the capture antibody is conjugated to a biotin moiety, the surface is coated with streptavidin, and the antibody is attached to the surface via a biotin-streptavidin bond, and / or the detection antibody binds to Aβ at a different epitope than the capture antibody, and / or the capture antibody comprises an affinity tag, and the detection antibody binds to the affinity tag, and / or the detectable label is a radioisotope, a chemiluminescent substance, a fluorescent substance, a metal complex, a bioluminescent substance such as luciferase, or a nucleic acid.

9. An antibody or antigen-binding fragment for use in a method for treating a neurological disorder associated with the accumulation of oAβ in a subject, the antibody or antigen-binding fragment comprising the antibody or antigen-binding fragment of claim 1, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment to the subject.

10. 10. The antibody or antigen-binding fragment of claim 9, wherein the disorders are cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD).

11. 10. The antibody or antigen-binding fragment of claim 9, administered to the brain of the subject.

12. 10. The antibody or antigen-binding fragment of claim 9, wherein the antibody is a monoclonal antibody, a humanized antibody, or a chimeric antibody.

13. A synthetic peptide immunogen for stimulating antibody development, which is specific for a conformational epitope present in aggregated forms of amyloid beta protein (Aβ), including multimers of amyloid beta protein (oAβ), but is not specific for the monomeric form.

14. 14. The synthetic peptide immunogen of claim 13, comprising the isolated amyloid beta sequence GYEVHHQKLV (SEQ ID NO: 1) or the sequence YEXHH (SEQ ID NO: 41), wherein X is a hydrophobic amino acid (V, I, or L).

15. The peptide comprises the peptide sequence CGKCGYEVHHQKLVVPNVLKQHHVEYGCK (SEQ ID NO: 2) or CGKCGYEVHHQKLVVPNGYEVHHQKLVCK ​​(SEQ ID NO: 3), or one of the peptide sequences SEQ ID NOs: 5-17 shown in Table A, and / or the peptide further comprises a carrier protein linked to its N-terminus, optionally wherein the carrier protein is linked by a maleimide, and / or the carrier protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or 14. The synthetic peptide of claim 13, wherein the carrier protein optionally comprises ovalbumin (OVA), and / or the carrier protein optionally comprises (i) a peptide having Pam3Cys(N-α-palmitoyl-S-2,3-bis(palmitoyloxy)-(2RS)-propyl-L-cysteine, with palmitoyl-Cys((RS)-2,3) attached to the second lysine (K4), with the sequence KSSKSKKKFISEAIIHHLHSRHPGK (SEQ ID NO: 4), wherein the carrier protein is conjugated to the N-terminus via a maleimide.

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