Diagnostic use of highly toxic amyloid oligomers

Abstract

Use of a novel highly toxic amyloid oligomer A [beta] o * 3F as a target for diagnosis of early and mid-advanced Alzheimer's disease (AD) and AD-derived mild cognitive impairment (MCI), A [beta] o * 3F is specifically bound by a 3F antibody, and exists in cerebrospinal fluid (CSF), blood and / or brain tissue of AD patients and AD-derived MCI patients, the Abeta oligomers have the advantages that the Abeta oligomers are high-toxicity oligomers, the levels of the Abeta oligomers are remarkably different in CSF, blood and / or brain tissues of AD patients, MCI patients and healthy old people, and the Abeta oligomers are super-toxicity oligomers, are the most major toxic components in A beta oligomer mixtures, have strong pathogenic effects and play a key role in occurrence and development of AD.

Description

Diagnostic Use of Highly Toxic Amyloid Oligomers Technical Field

[0001] The present invention relates to the diagnostic use of highly toxic amyloid oligomers. Specifically, the present invention relates to the use of the novel highly toxic amyloid oligomer Aβo*3F as a target for diagnosing early and mid-to-late stage Alzheimer's disease (AD) and AD-induced mild cognitive impairment (MCI). Background Art

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disorder that affects approximately 50 million people worldwide. To date, there are no specific, effective treatments, placing a heavy burden on humanity. AD progresses over a long period of time, initially asymptomatic, gradually developing into memory loss and personality and behavioral changes. In later stages, AD leads to widespread neuronal death and significant brain atrophy, making it difficult for therapeutic drugs to be effective. Clinical trials in recent years have demonstrated that early intervention in patients with AD, identified early and with early warning signs, can slow its progression. However, achieving early treatment and intervention requires timely and accurate identification of AD patients, but currently, no ideal early diagnosis methods or technologies are available in clinical practice. Studies have shown that changes in amyloid-β (Aβ), which trigger the development of AD, can occur 15 to 20 years before clinical symptoms appear.

[0003] The pathological hallmarks of AD are senile plaques formed by the aggregation of Aβ and neurofibrillary tangles formed by the aggregation of tau proteins in the brain. Aβ can aggregate into oligomers, profibrils, and mature fibrils. Aβ oligomers are the most neurotoxic aggregate form, with Aβ dimers, trimers, and ADLs being the most commonly reported. Aβ oligomers can be divided into low-molecular-weight and high-molecular-weight types based on their molecular weight, with high-molecular-weight oligomers exhibiting greater neurotoxicity. Aβ dimers are the smallest Aβ oligomers and are generally considered to be the basic building block of Aβ oligomers. Aβ dimers are found at elevated levels in the brains of AD patients and AD transgenic mice, are stable in SDS and strong denaturants, and possess a degree of neurotoxicity. In addition to Aβ dimers, Aβ trimers are also considered to be the aggregated units of various Aβ oligomers, such as hexamers and dodecamers. Aβ trimers appear early in the brains of AD patients and AD transgenic mice, but there is no significant correlation between Aβ trimers and Aβ plaque deposition, and their toxicity remains controversial. In addition, Aβ oligomers can also further aggregate to form spherical oligomers (ASPD) with a diameter of about 12 nm and diffusible oligomers (ADDL) with a diameter of 5-6 nm. Both forms of polymers are considered to be Aβ oligomers with unique conformations and neurotoxicity. At present, although a variety of Aβ oligomer forms have been discovered, it is still unknown which Aβ oligomer or oligomers are the most toxic and play a major role in the occurrence and development of AD. Moreover, the methods for specifically detecting oligomers, especially toxic oligomers, are very limited, and it is particularly difficult to obtain antibodies that specifically bind to toxic amyloid protein oligomers, which seriously limits the clinical detection and development of therapeutic preparations for Aβ oligomers.

[0004] Numerous studies have shown that the severity of neurodegenerative diseases is closely correlated with the level of amyloid oligomers in the patient's brain. Aβ oligomers play a key role in the development and progression of AD by causing functional neuronal death, cognitive impairment, and dementia. However, Aβ oligomers are diverse and can affect central nervous system function through a variety of mechanisms. They also exhibit varying toxicity, with significant differences in size, conformation, aggregation pattern, toxicity, and the timing of their appearance in the brain. Although numerous studies have demonstrated that some Aβ oligomers, such as dimers, trimers, and ADDLs, can exert neurotoxic effects, the precise form of Aβ oligomer that truly plays a key pathogenic role remains largely unknown. Therefore, identifying key toxic oligomers closely associated with the development and progression of AD could provide ideal biomarkers for early warning and diagnosis of AD, as well as promising targets for its treatment.

[0005] In addition to imaging, specific biomarkers are fundamental to the clinical diagnosis and testing of AD. Currently, the most widely accepted AD diagnostic markers are Aβ42, Aβ40, total tau (T-Tau), and phosphorylated tau (P-Tau) in cerebrospinal fluid (CSF). Other marker proteins have also been reported, such as sAPPα, sAPPβ, BACE1, Aβ oligomers, total Aβ, and axonal and synaptic markers. These CSF markers have high diagnostic accuracy and, when combined with other AD diagnostic indicators, can achieve a diagnostic sensitivity and specificity of 85-90%. Compared to CSF, blood is easily accessible and less invasive, making it an ideal sample source for clinical testing. Several studies have begun to use a combination of peripheral blood protein, lipid, and metabolite levels to distinguish AD patients from healthy controls. These include the levels of Aβ42 dimers on peripheral blood cell membranes, plasma gelsolin (GSN), which disaggregates Aβ42 fibrils, and MMP3, the primary GSN degrading enzyme. Recently, the detection of specific components in peripheral blood, such as Tau181, Tau217, and Aβ content, has also become a research hotspot.

[0006] Mainstream biochemical testing methods for AD primarily focus on measuring Aβ and Tau protein markers in CSF. Numerous studies have shown that as healthy individuals progress to early cognitive impairment (MCI) and AD, CSF levels of Aβ42 monomers gradually decrease, while levels of Aβ42 oligomers and T-Tau gradually increase, with the P-Tau to Aβ42 ratio changing more significantly. However, obtaining CSF samples requires a lumbar puncture, which is quite invasive and often difficult for patients with MCI and mild AD to undergo. Furthermore, the lack of standardized CSF sample pretreatment and testing methods leads to significant discrepancies in test results between different manufacturers and laboratories, severely limiting the clinical application of these methods. Compared to CSF, blood-based testing offers lower costs and higher compliance. However, the low levels and limited variety of markers available for AD diagnosis in blood, coupled with significant influence from peripheral tissues and organs, have hindered the development of AD diagnostic methods. Currently, the most advanced methods internationally involve measuring the ratio of Aβ42 to Aβ40 in blood, as well as the levels of proteins such as P-Tau181 and P-Tau217, using liquid chromatography-mass spectrometry (LC-MS) and single-molecule immunoassay technology (Simoa). While these blood markers have some diagnostic value, they lack specificity, and single markers cannot effectively differentiate target populations. This is because the detection of Aβ or phosphorylated Tau protein measures the total amount, which includes monomers and their various aggregates with varying toxicities. Monomers and many types of oligomers have low cytotoxicity, but their high abundance contributes significantly to overall Aβ levels, significantly interfering with the detection of the more toxic amyloid oligomers that play a key role in the pathogenesis of AD. However, only highly toxic oligomers are directly associated with the development and progression of AD. Their presence and levels directly and accurately correlate with brain pathology in patients, making specific detection of these oligomers more valuable and meaningful.

[0007] Therefore, there is still an urgent need in the art to perform high-sensitivity and high-specificity detection of specific Aβ oligomer content in the blood for diagnosing AD.

[0008] Summary of the Invention

[0009] One aspect of the present invention relates to a method for diagnosing whether a subject has early-stage or mid-stage AD or AD-induced MCI or is at risk of having AD, the method comprising the steps of:

[0010] a) optionally, providing a sample to be tested from a subject,

[0011] b) contacting a sample from the subject with a reagent for detecting the presence and / or level of Aβo*3F in the sample from the subject,

[0012] c) detecting the presence and / or level of Aβo*3F in a sample from the subject,

[0013] The presence and / or level of Aβo*3F in the subject sample indicates that the subject suffers from AD or AD-induced MCI, wherein the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody, and based on size exclusion chromatography (SEC) analysis, its molecular weight is approximately 588 kDa, and the light and heavy chain CDR sequences of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively.

[0014] In some embodiments, the subject is a patient suspected of having AD or AD-induced MCI, preferably, the subject is a human, a non-human primate, a cat, or a dog.

[0015] In some embodiments, the diagnostic method further comprises clinical neuropsychological and neuroimaging assessment steps, preferably, the neuroimaging is Aβ-PET scanning imaging and / or tau-PET. More preferably, the neuroimaging is 3F-PET scanning imaging, i.e., 3F-based immunoPET imaging, for identifying and detecting the presence and / or level of Aβo*3F in the subject's brain.

[0016] In some embodiments, the diagnostic method further comprises detecting changes in tau levels in the subject's sample. Preferably, the tau level is the total tau level or the phosphorylated tau level.

[0017] The second aspect of the present invention relates to a kit for diagnosing whether a subject has early-stage or mid-late-stage AD or AD-induced MCI or is at risk of developing AD, comprising a reagent for detecting the presence and / or level of Aβo*3F in a sample from the subject, wherein the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody, and based on size exclusion chromatography analysis, its molecular weight is approximately 588 kDa, and the light and heavy chain CDR sequences of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively.

[0018] In some embodiments, the subject sample is a sample obtained from the subject on-site when performing the diagnosis. In some embodiments, the subject sample is a sample obtained from the subject in a previous diagnostic procedure.

[0019] In some embodiments, the size exclusion chromatography analysis is performed using a Superdex 200 10 / 300GL molecular sieve chromatography column.

[0020] In some embodiments, Aβo*3F is present in a cerebrospinal fluid sample obtained from a subject. In some embodiments, the cerebrospinal fluid sample from the subject is analyzed using immunoprecipitation and size exclusion chromatography to determine the molecular weight of Aβo*3F.

[0021] In some embodiments, the method or kit involves or includes an Aβo*3F enrichment reagent, the Aβo*3F enrichment reagent including a binding agent that specifically binds to Aβo*3F, preferably, the binding agent that specifically binds to Aβo*3F is an antibody that specifically binds to Aβo*3F, preferably, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof, more preferably, the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, diabody, or bispecific antibody. By using the Aβo*3F enrichment reagent, Aβo*3F in a subject's sample can be effectively enriched for use in subsequent procedures.

[0022] In some embodiments, the reagent for detecting the presence and / or level of Aβo*3F includes a first binding agent that specifically binds to Aβo*3F or Aβ aggregates. Preferably, the first binding agent that specifically binds to Aβo*3F or Aβ aggregates is an antibody that specifically binds to Aβo*3F or Aβ aggregates. Preferably, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof. More preferably, the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, diabody, or bispecific antibody. In some embodiments, the first binding agent that specifically binds to Aβo*3F or Aβ aggregates is a scFv or full-length antibody that specifically binds to Aβo*3F or Aβ aggregates.

[0023] In some embodiments, the first binding agent is linked to a detection agent that allows for its detection.

[0024] In some embodiments, the Aβo*3F can be detected and defined using the K98R mutant of the 3F antibody. The Aβ oligomers detected using the K98R mutant are consistent with the Aβ oligomers detected by the 3F antibody, both of which are Aβo*3F. However, the K98R mutant has a higher affinity for binding to Aβo*3F than 3F, and can more sensitively detect the presence or absence of Aβo*3F and can more sensitively diagnose neurodegenerative diseases such as AD.

[0025] In some embodiments, the first binding agent that specifically binds Aβo*3F or Aβ aggregates is the 3F antibody or a K98R mutant thereof.

[0026] In some embodiments, the reagent for detecting the presence and / or level of Aβo*3F further comprises a second binding agent that specifically binds to the first binding agent. Preferably, the second binding agent is an antibody that specifically binds to the first binding agent. Preferably, the antibody that specifically binds to the first binding agent is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof. More preferably, the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camelid antibody, nanobody, diabody, or bispecific antibody. Preferably, the antibody that specifically binds to the first binding agent is linked to a detection agent that allows its detection. In some embodiments, the antibody that specifically binds to the first binding agent is a full-length antibody.

[0027] In some embodiments, the detection agent is selected from the group consisting of a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a fluorescein-type label, an umbelliferone, a lissamine, a cyanine, a Texas Red, a BODIPY (Invitrogen) or its analogs, paramagnetic labels, radioactive labels, biotin, streptavidin / biotin, avidin / biotin, haptens, digoxigenin, metal complexes, metals, enzymes, colloidal gold, or combinations thereof. In some embodiments, the detection agent is selected from a chemiluminescent label, an electrochemiluminescent label, a chromophore, a fluorescent label, a radioactive label, an enzyme, or a combination thereof. In some embodiments, the detection agent is a chemiluminescent label or an electrochemiluminescent label.

[0028] In some embodiments, the detection is selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology (Simoa), flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot, protein chip, positron emission tomography and / or single photon emission computed tomography. Preferably, the kit includes reagents, materials, containers and / or devices required for performing the detection selected from chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology, flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot and / or protein chip. Preferably, the enzyme-linked immunosorbent assay is selected from direct enzyme-linked immunosorbent assay, indirect enzyme-linked immunosorbent assay, direct sandwich enzyme-linked immunosorbent assay and indirect sandwich enzyme-linked immunosorbent assay.

[0029] In some embodiments, the subject sample is selected from cells, tissues, organs and / or body fluids of the subject, preferably, the body fluid is selected from whole blood, plasma, serum, cerebrospinal fluid, lymph, saliva, synovial fluid, bronchoalveolar lavage fluid, sputum, ascites, urine, amniotic fluid, peritoneal fluid, pericardial fluid, semen and / or vaginal secretions, preferably, the body fluid is selected from whole blood, plasma, serum and / or cerebrospinal fluid.

[0030] In some embodiments, the enrichment agent or first binding agent that specifically binds Aβo*3F is attached to a solid support.

[0031] In some embodiments, the method further comprises the step of detecting a control sample, and / or the kit further comprises a control sample, wherein the control sample is from a healthy subject or a subject not suffering from AD and AD-induced MCI.

[0032] In some embodiments, the antibody that specifically binds to Aβo*3F is a polyclonal antibody and / or monoclonal antibody or an antigen-binding fragment thereof obtained by immunization against Aβo*3F. Preferably, the antibody is a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a sheep antibody or a non-human primate antibody.

[0033] In some embodiments, based on the MSD electrochemiluminescence method, the concentration of Aβo*3F in the cerebrospinal fluid of healthy subjects is Aβ42o*3F: 80.44±20.88 pg / ml, Aβ40o*3F: 24.35±5.08 pg / ml; and / or the concentration of Aβo*3F in plasma is Aβ42o*3F: 71.63±36.8 pg / ml, Aβ40o*3F: 2.24±0.92 pg / ml; the concentration of Aβo*3F in the plasma of MCI subjects is Aβ42o*3 F: 112.93 ± 25.02 pg / ml, Aβ40o*3F: 4.64 ± 1.43 pg / ml; the concentration of Aβo*3F in the cerebrospinal fluid of AD patients is Aβ42o*3F: 264.8 ± 42.26 pg / ml, Aβ40o*3F: 85.74 ± 10.62 pg / ml, and / or the concentration of Aβo*3F in plasma is Aβ42o*3F: 159.44 ± 36.8 pg / ml, Aβ40o*3F: 14.0 ± 5.59 pg / ml; or

[0034] Based on the chemiluminescence method, the concentration of Aβ42o*3F in the plasma of healthy subjects was 68.02±39.17 pg / ml; the concentration of Aβ42o*3F in the plasma of patients with MCI was 124.5±12.57 pg / ml; and the concentration of Aβ42o*3F in the plasma of patients with AD was 205.75±50.96 pg / ml. In some embodiments, the MSD electrochemiluminescence method is performed as described in Example 7 of the specification. In some embodiments, the chemiluminescence method is performed as described in Example 8 of the specification.

[0035] In some embodiments, the detection sensitivity is as low as 0.5 pg / ml based on MSD electrochemiluminescence method and chemiluminescence method.

[0036] In some embodiments, the antibody that specifically binds to Aβo*3F is a plurality of antibodies specific for different epitopes of Aβo*3F, for example, 2, 3, 4, 5 or more antibodies that are respectively specific for 2, 3, 4, 5 or more different epitopes of Aβo*3F.

[0037] In some embodiments, the step of enriching Aβo*3F from the sample is performed using immunoprecipitation with an antibody that specifically binds to Aβo*3F.

[0038] In other words, in previous studies, the inventors used phage display technology to be the first in the world to screen out a fully human single-chain antibody W20 that specifically binds to Aβ oligomers (see CN101463082A). The 3F antibody (amino acid sequence is shown in SEQ ID No. 23) is an improved form of the W20 antibody. Compared with the W20 antibody, it has significantly improved affinity for Aβ oligomers, can more significantly inhibit the aggregation of Aβ and the neurotoxicity induced by Aβ oligomers, more effectively improve the cognitive and memory functions of AD model mice, and reduce pathological changes in the mouse brain. More significantly, the Aβ oligomers specifically recognized by this antibody are highly toxic oligomers, the most predominant toxic component of the Aβ oligomer mixture. They possess potent pathogenicity and play a key role in the development and progression of AD. The highly toxic oligomers recognized by 3F are present in the CSF, blood, and / or brain tissue of AD patients and patients with AD-induced MCI. Their levels are highly variable in the CSF, blood, and / or brain tissue of AD patients, MCI patients, and healthy elderly individuals, enabling precise differentiation of these three groups. These toxic oligomers are also present in AD transgenic mice and are directly associated with their pathogenesis. The highly toxic Aβ oligomers identified by 3F are referred to in this invention as AβO*3F. They can be isolated from a mixture of Aβ oligomers by immunoprecipitation (using the 3F antibody). They are typically characterized as high-molecular-weight Aβ oligomers, with a molecular weight of approximately 588 kDa and a diameter of approximately 10 nm, as determined by size exclusion chromatography (SEC). They are highly toxic to neurons, with toxicity exceeding 200 times that of the Aβ oligomer mixture. Aβo*3F can activate microglia and astrocytes, leading to the secretion of large amounts of inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows a schematic diagram of immunoprecipitation preparation of Aβo*3F, Aβ*6E10, and Aβ-ID.

[0040] Figure 2 shows the molecular weight and morphology of Aβo*3F:

[0041] A: ThT detects Aβ aggregation status;

[0042] B: Dot blot analysis of the binding of oligomers obtained under different incubation conditions to 3F, A11, and 6E10 antibodies;

[0043] C: Affinity of Aβos obtained by incubation with 10 μM Aβ for 0-4 days and 3F antibody;

[0044] D: Western blot detection of sAβo*3F band distribution;

[0045] E: Size exclusion chromatography (SEC) analysis of sAβo*3F molecular weight; SEC analysis (Superdex 200 10 / 300) of sAβo*3F and SEC standards. SEC standards: 1. thyroglobulin (669 kDa), 2. ferritin (440 kDa), 3. aldolase (158 kDa), 4. conalbumin (75 kDa), 5. ovalbumin (44 kDa), 6. carbonic anhydrase (29 kDa).

[0046] F: linear equation (Y) and correlation coefficient (r2) based on the molecular weight and elution volume of the SEC standard;

[0047] G: Western blot detection of mAβo*3F band distribution;

[0048] H: SEC analysis of the molecular weight of mAβo*3F;

[0049] I: Western blot detection of hAβo*3F band distribution.

[0050] Figure 3 shows the neurotoxic effects of Aβo*3F:

[0051] A: IC50 of sAβos cytotoxicity to N2a cells;

[0052] B: IC50 of sAβo*3F cytotoxicity to N2a cells;

[0053] C: MTT assay to compare the neurotoxicity of sAβos, sAβo*3F and sAβ-ID on N2a cells;

[0054] D: IC50 of mAβo*3F toxicity to N2a cells and primary neurons;

[0055] E: MTT assay to compare the cytotoxicity of mAβ*6E10, mAβo*3F and mAβ-ID on primary neurons;

[0056] F: IC50 of hAβo*3F toxicity to primary neurons;

[0057] G: MTT assay was used to compare the cytotoxicity of hAβ*6E10, hAβo*3F and hAβ-ID on primary neurons.

[0058] Figure 4 shows the effects of Aβo*3F on cytokine expression levels in glial cells:

[0059] A: Effects of mAβo*3F on the expression levels of TNF-α, IL-1β, and IL-6 in microglia;

[0060] B: Effects of mAβo*3F on the expression levels of TNF-α, iNos, IL-1β, and IL-6 in primary astrocytes;

[0061] C: Effect of mAβo*3F on the expression levels of TSP1, Gpc4 and Gpc6 in primary astrocytes.

[0062] Figure 5 shows the effects of Aβo*3F on mouse cognition and its damaging effects on brain neurons:

[0063] A: Cognitive index of mice in each group in the novel object recognition experiment;

[0064] B: The duration of each group of mice in the novel arm in the Y maze test;

[0065] C: Golgi staining to detect the density of dendritic spines in mouse neurons;

[0066] D: Statistical analysis of dendritic spine density in C;

[0067] E: Nissl staining to detect the number of neurons in the mouse hippocampus, scale bar: 20 μm;

[0068] F, G: Statistical analysis of the number of neurons in the DG region (F) and CA1 region (G) was performed using Image J software.

[0069] Figure 6 shows that Aβo*3F activates glial cells in the mouse brain to produce neuroinflammation:

[0070] A: Immunohistochemistry assay to detect the activation of microglia in the DG and CA1 regions of the mouse hippocampus. Scale bar: 20 μm.

[0071] B: Immunohistochemistry assay to detect the activation of astrocytes in the DG and CA1 regions of the mouse hippocampus. Scale bar: 20 μm.

[0072] C, D: The area of ​​Iba-1-positive microglia in the DG (C) and CA1 (D) regions was quantified using Image J software;

[0073] E, F: The area of ​​GFAP-positive astrocytes in the DG (E) and CA1 (F) regions was quantified using Image J software;

[0074] G, H: ELISA was used to detect the expression levels of IL-6 (G) and IL-1β (H) in the hippocampus of mice.

[0075] Figure 7 shows the levels of Aβo*3F in CSF and plasma of AD patients detected by electrochemiluminescence: A, B: MSD detection of the corresponding levels of Aβ42 (A) and Aβ40 (B) in Aβo*3F isolated from CSF of AD patients and healthy subjects (Con);

[0076] C, D: MSD method was used to detect the corresponding Aβ42 (C) and Aβ40 (D) levels in Aβo*3F isolated from the plasma of patients with mild cognitive impairment (MCI), AD and healthy subjects (Con).

[0077] Figure 8 shows the chemiluminescence detection of Aβo*3F levels in the plasma of AD patients, MCI patients and healthy subjects.

[0078] FIG9 shows the use of Aβo*3F levels in CSF or plasma samples for AD diagnosis.

[0079] Figure 10 shows that after the K at position 98 of the 3F antibody heavy chain mutated to R, the affinity for binding to Aβo*3F oligomers was further improved, suggesting that the optimized single-chain antibody can be used to better diagnose the presence of Aβo*3F oligomers and thus to better diagnose AD. DETAILED DESCRIPTION

[0080] definition

[0081] Unless otherwise indicated, the terms used in the claims and specification are defined as shown below.

[0082] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the methods and compositions of the present invention as described herein shall have the meanings commonly understood by those of ordinary skill in the art. Additionally, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Typically, the nomenclature used in conjunction with the following techniques are those well known and commonly used in the art: biochemistry, immunology, enzymology, molecular and cellular biology, microbiology, genetics, and polypeptide chemistry as described herein.

[0083] The methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplements through 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, NJ; Handbook of Biochemistry: Section A Proteins, Vol. I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol. II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

[0084] All publications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety.

[0085] Unless otherwise indicated, the following terms shall be understood to have the following meanings.

[0086] The term "at risk of developing Alzheimer's disease" refers to having a risk of developing Alzheimer's disease, such as family history, aging, unhealthy lifestyle such as drinking, and the like.

[0087] The term "Aβ-PET scan" refers to positron emission tomography of beta-amyloid.

[0088] The term "tau-PET scan" refers to positron emission tomography of the tau protein.

[0089] The term "3F-PET imaging" refers to immunopositron emission tomography using the 3F antibody. The term "Aβo*3F" refers to Aβ oligomers that specifically bind to the 3F antibody, having a molecular weight of approximately 588 kDa as determined by size exclusion chromatography (SEC). The light and heavy chain CDR sequences of the 3F antibody are set forth in SEQ ID NOs: 17-22, respectively.

[0090] The term "Aβ42o*3F" refers to the Aβ42 oligomer portion in Aβo*3F.

[0091] The term "Aβ40o*3F" refers to the Aβ40 oligomer portion of Aβo*3F.

[0092] In some embodiments, the chemiluminescent labels of the present disclosure are selected from alkaline phosphatase, horseradish peroxidase, isoluminol and its derivatives, acridinium esters and their derivatives.

[0093] In some embodiments, the chromophores of the present disclosure are selected from ruthenium terpyridine.

[0094] In some embodiments, the fluorescent labels of the present disclosure are selected from rare earth chelates or derivatives thereof, isothiocyanates, rhodamine, phycobilin, phycoerythrin, phycocyanin, allophycocyanin, fluorescein isothiocyanate, or AlexaFluor dyes.

[0095] In some embodiments, the luciferin-type labels of the present disclosure are selected from luciferin, luciferase, such as firefly luciferase, and bacterial luciferase.

[0096] In some embodiments, the paramagnetic label includes, but is not limited to, aluminum (Al), barium (Ba), calcium (Ca), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (Mo), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), platinum (Pt), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W), vanadium (V), and zirconium (Zi), and in particular Co. +2 , CR +2 Cr +3 、Cu +2 、Fe +2 、Fe +3 、Ga +3 、Mn +3 、Ni+2 、Ti +3 、V +3 and V +4 Compounds containing paramagnetic ions.

[0097] In some embodiments, the radiolabel is selected from technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulfur-35, deuterium, tritium, rhenium-186, rhenium-188, and yttrium-90.

[0098] In some embodiments, the hapten is selected from a polysaccharide or a lipid.

[0099] In some embodiments, the metal complex is selected from ruthenium terpyridine.

[0100] In some embodiments, the metal is selected from ruthenium.

[0101] In some embodiments, the enzyme of the present disclosure is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, β-glucuronidase, β-D-glucosidase, urease, hexokinase, malic enzyme, glucose-6-phosphate dehydrogenase, sucrase, glucoamylase, lysozyme, carbohydrate oxidase, heterocyclic oxidase, streptavidin-β-D-galactopyranose conjugate and / or streptavidin-horseradish peroxidase conjugate.

[0102] In some embodiments, ruthenium terpyridine is used for electrochemiluminescence assays.

[0103] In some embodiments, the detection agent for the biotinylated detection antibody is avidin, streptavidin-HRP, or streptavidin-β-D-galactopyranose (SBG). In certain embodiments, the readout of the detection agent is fluorometric or colorimetric. For example, but not limited to, tetramethylbenzidine and hydrogen peroxide can be used as the readout. In certain embodiments, if the detection agent is streptavidin-HRP, the readout can be colorimetric using tetramethylbenzidine and hydrogen peroxide. Alternatively, in certain embodiments, resorufin β-D-galactopyranoside can be used as the readout. For example, but not limited to, if the detection agent is SBG, the readout can be fluorometric using resorufin β-D-galactopyranoside.

[0104] In certain embodiments, the detection agent, such as SBG, can be used at a concentration of about 50 to about 500 pM. For example, but not limited to, the detection agent can be used at a concentration of about 50 to about 100 pM, about 50 to about 150 pM, about 50 to about 200 pM, about 50 to about 250 pM, about 50 to about 300 pM, about 50 to about 350 pM, about 50 to about 400 pM, about 50 to about 450 pM, about 100 to about 500 pM, about 150 to about 500 pM, about 200 to about 500 pM, about 250 to about 500 pM, about 300 to about 500 pM, about 350 to about 500 pM, about 400 to about 500 pM, about 450 to about 500 pM, about 100 to about 400 pM, or about 200 to about 400 pM. In certain embodiments, the detection agent can be used at a concentration of about 100 pM to about 400 pM. For example, SBG can be used at a concentration of about 110 pM, about 155 pM, or about 310 pM. In certain embodiments, SBG can be used at a concentration of about 310 pM. In certain embodiments, the detection agent, such as HRP, can be used at a dilution of about 1 / 10 to about 1 / 1000. For example, but not limited to, the detection agent can be used at a dilution of about 1 / 10 to about 1 / 100, about 1 / 10 to about 1 / 500, about 1 / 100 to about 1 / 1000, or about 1 / 500 to about 1 / 1000. In certain embodiments, the detection agent can be used at a dilution of about 1 / 100 to about 1 / 1000. For example, HRP can be used at a dilution of about 1 / 100 or about 1 / 500. The concentration of the detection agent to be used can be readily determined by one skilled in the art, depending on the type of detection agent, without exceeding routine experimentation.

[0105] In some embodiments, as an enzyme color development matrix, for example, when horseradish peroxidase (HRP) is selected as an enzyme label, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, anisidine or 3,3'-dimethoxybenzidine can be used as a matrix. Also, when alkaline phosphatase is selected as an enzyme label, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitro blue tetrazolium or p-nitrophenol phosphate can be used as a matrix. Furthermore, when β-D-glucosidase is selected as the enzyme label, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside can be used as a substrate. In addition, various enzymes and enzyme color development substrates known in the art can be used.

[0106] In certain embodiments, the methods of the present disclosure may include blocking the first binding agent with a blocking buffer. In certain embodiments, the blocking buffer may include PBS, bovine serum albumin (BSA), and / or a biocide, such as ProClin TM (Sigma-Aldrich, Saint Louis, MO). In certain embodiments, the method can include multiple washing steps. In certain embodiments, the solution used for washing is typically a buffer (e.g., a "wash buffer"), such as, but not limited to, PBS buffer including a detergent (e.g., Tween 20). For example, but not limited to, the first binding agent can be washed after blocking and / or the sample can be separated from the first binding agent (e.g., by washing) to remove unbound material.

[0107] In some embodiments, the diagnostic methods of the present invention have a detection sensitivity as low as about 0.5 pg / mL, for example, about 1.0 pg / mL, 1.5 pg / mL, 2.0 pg / mL, 2.5 pg / mL, 3.0 pg / mL, 3.5 pg / mL, 4.0 pg / mL, 4.5 pg / mL, 5.0 pg / mL, 5.5 pg / mL, 6.0 pg / mL, 7.5 pg / mL, 8.0 pg / mL, 8.5 pg / mL, 9.0 pg / mL, g / mL, 9.5pg / mL, 10.0pg / mL, 15pg / mL, 20pg / mL, 25pg / mL, 30pg / mL, 50pg / mL, 100pg / mL, 150pg / mL, 200 pg / mL, 250pg / mL, 500pg / mL, 750pg / mL, 1000pg / mL, 1500pg / mL, 2000pg / mL, 3000pg / mL, 5000pg / mL or higher.

[0108] In some embodiments, the kits of the present invention include a first container comprising an enrichment reagent or a first binding agent that specifically binds to Aβo*3F.

[0109] In some embodiments, kits of the invention include a second container comprising a second binding agent that specifically binds to the first binding agent.

[0110] The term "a second binding agent specifically binds to the first binding agent" refers to the antibody class to which the second binding agent specifically binds, such as an antibody. For example, if the first binding agent is mouse anti-Aβo*3F IgG, the second binding agent can be rabbit anti-mouse IgG.

[0111] In some embodiments, the kits of the invention include a third container containing a control sample.

[0112] In some embodiments, the kit of the present invention includes one or more other containers containing other reagents and materials required for performing the detection.

[0113] In some embodiments, the enrichment agent or first binding agent that specifically binds to Aβo*3F, or the second binding agent that specifically binds to the first binding agent, is attached to a solid support. In some embodiments, the solid support is selected from polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, plasma substitute, cross-linked glucose, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamide, polystyrene, gypsum, filter paper, ion exchange resin, plastic film, plastic tube, poly(methyl vinyl ether / maleic acid) copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or magnetic particles. Other solid supports include cell culture plates, enzyme-linked immunosorbent assay plates, electrochemiluminescence assay plates, tubes, and polymeric membranes. The solid support can have any form, such as spherical (microbeads), cylindrical (inside a test tube or well), or flat (sheet, test strip).

[0114] In some embodiments, the kits disclosed herein are kits for performing an enzyme-linked immunosorbent assay (ELISA), comprising some or all of the reagents, materials, containers, and / or apparatus required for performing an ELISA. Those skilled in the art will appreciate how to prepare corresponding ELISA kits based on the disclosed reagents for detecting the presence and / or level of Aβo*3F in a subject's sample. In some embodiments, the ELISA kits comprise a pre-coated Aβo*3F antibody, an HRP-labeled Aβ antibody, a standard, a wash solution, a substrate, and the like.

[0115] In some embodiments, the kit of the present disclosure is a kit for performing an immunofluorescence assay, which includes some or all of the reagents, materials, containers, and / or devices required for performing an immunofluorescence assay. Those skilled in the art will know how to prepare a corresponding immunofluorescence assay kit based on the reagents disclosed herein for detecting the presence and / or level of Aβo*3F in a subject sample. In some embodiments, the immunofluorescence assay kit is an electrochemiluminescent immunoassay. In some embodiments, the immunofluorescence assay kit is a time-resolved immunofluorescence assay. In some embodiments, the immunofluorescence assay kit includes an electrochemiluminescent plate pre-coated with an Aβo*3F antibody, a ruthenium-labeled Aβ antibody, a standard, a read buffer, and the like.

[0116] In some embodiments, the kit disclosed herein is a kit for performing a colloidal gold assay, comprising some or all of the reagents, materials, containers, and / or apparatus required for performing a colloidal gold assay. Those skilled in the art will appreciate how to prepare a corresponding colloidal gold assay kit based on the disclosed reagents for detecting the presence and / or level of Aβo*3F in a subject's sample. In some embodiments, the colloidal gold assay kit is a colloidal gold test strip. In some embodiments, the colloidal gold assay kit comprises a colloidal gold-labeled Aβo*3F antibody, an Aβ antibody, a control antibody, and the like.

[0117] In some embodiments, the kits disclosed herein are kits for performing radioimmunoassays, comprising some or all of the reagents, materials, containers, and / or apparatus required for performing a radioimmunoassay. Those skilled in the art will appreciate how to prepare corresponding radioimmunoassay kits based on the reagents disclosed herein for detecting the presence and / or level of Aβo*3F in a subject's sample. In some embodiments, the radioimmunoassay kits comprise radioisotope-labeled Aβo*3F antibodies, Aβo*3F standards, and the like.

[0118] The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule that has the ability to bind to an epitope on an antigen. Naturally occurring antibodies typically comprise tetramers, which are generally composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the following isotypes: IgG, IgA, IgM, IgD, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Igs of the same class are further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of disulfide bonds in their heavy chains. For example, IgG is divided into IgG1, IgG2, IgG3, and IgG4 subclasses, and IgA is divided into IgA1 and IgA2 subclasses. Light chains are divided into kappa and lambda chains based on differences in their constant regions. The antibodies of the present invention may have any isotype. The choice of isotype is generally determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant domains κ or λ can be used. If necessary, the class of the antibody of the present invention can be converted by known methods. For example, an antibody of the present invention that is initially an IgG can be converted to an IgM antibody of the present invention by class conversion. In addition, class conversion technology can be used to convert one IgG subclass into another subclass, for example, from IgG1 to IgG2. Therefore, the effector function of the antibody of the present invention can be converted to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies by isotype switching for various therapeutic uses, provided that the C1q binding activity of the antibody is reduced or eliminated. In some embodiments, the antibody of the present invention is an IgM or IgG1, 2, 3 or 4 type antibody. If the amino acid sequence of the antibody is largely homologous to the isotype relative to other isotypes, the antibody belongs to a specific isotype.

[0119] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to complete antibodies and antigen-binding fragments of antibodies.

[0120] A "variable region" or "variable domain" is a domain in an antibody's heavy or light chain that participates in binding the antibody to its antigen. Each heavy chain of an antibody is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which is typically composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The heavy and light chain variable regions are typically responsible for antigen recognition, while the heavy and light chain constant domains can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), Fc receptors, and the first component (C1q) of the classical complement system. The heavy and light chain variable regions contain a binding region that interacts with an antigen. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) called "complementarity determining regions (CDRs)," which are interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDR domains and four FR domains, arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0121] The term "complementarity determining region" or "CDR region" or "CDR" (used interchangeably herein with hypervariable region "HVR") refers to the region in the antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigenic epitopes. In this article, the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0122] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0123] The term "monoclonal antibody", "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule preparation of a single molecular composition, which refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the group comprising individual antibodies is identical except for possible naturally occurring mutations that may be present in small amounts. Conventional monoclonal antibody compositions exhibit a single binding specificity and affinity for a specific epitope. In certain embodiments, a monoclonal antibody can be composed of more than one Fab domain, thereby increasing specificity for more than one target. The term "monoclonal antibody" or "monoclonal antibody composition" is not limited to any specific production method (e.g., recombinant, transgenic, hybridoma, etc.).

[0124] The present invention also includes "bispecific antibodies," wherein the antibodies of the present invention are part of a bivalent or multivalent bispecific framework that targets more than one epitope (e.g., the second epitope can comprise an epitope of an active transport receptor, such that the bispecific antibody exhibits improved transcytosis across biological barriers (e.g., the blood-brain barrier)). Thus, in another embodiment, a monovalent Fab of an antibody of the present invention can be linked to another Fab or scFv targeting a different protein to produce a bispecific antibody. Bispecific antibodies can have dual functions, such as a therapeutic function conferred by the present invention and a transport function that can bind to a receptor molecule to enhance translocation across a biological barrier (e.g., the blood-brain barrier). The terms "bispecific antibody," "bifunctional antibody," "bispecific antibody," "bispecific antibody," or "BsAb" refer to antibodies that possess two different antigen-binding sites, thereby being able to bind to two target antigens simultaneously, mediating another specific function while exerting the antibody's targeting properties. The specific function effector molecule mediated can also be a toxin, enzyme, cytokine, radionuclide, etc. The two arms of the bispecific antibody that bind to the antigen can be derived from Fab, Fv, ScFv, or dSFv, etc.

[0125] The term "antigen-binding fragment of an antibody" refers to a fragment, part, region or domain of an antibody that can bind to an epitope (e.g., obtainable via cleavage, recombination, synthesis, etc.). An antigen-binding fragment may contain 1, 2, 3, 4, 5 or all 6 CDR domains of such an antibody, and despite being able to bind to the epitope, may exhibit different specificities, affinities or selectivities. Preferably, the antigen-binding fragment contains all 6 CDR domains of the antibody. An antigen-binding fragment of an antibody may be a part of or comprise a single polypeptide chain (e.g., scFv), or may be a part of or comprise two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., diabodies, Fab fragments, F(ab')2 fragments, etc.).

[0126] Examples of antigen-binding fragments encompassed by the present invention include (a) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (b) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge domain; (c) Fd fragments consisting of the VH region and the CH1 domain; (d) Fv fragments consisting of the VL and VH regions of a single antibody arm; (e) single-chain antibodies (scFv), recombinant proteins formed by genetically engineering the VH and VL regions of an antibody linked by a linker peptide; (f) dAb fragments (Ward et al., Nature, 341, 544-546 (1989)), which consist essentially of the VH region and are also known as domain antibodies (Holt et al., Trends Biotechnol., 2i(11):484-90); (g) camelid or nanobodies (Revets et al., Expert Opin Biol., 21(11):509-610); Ther., 5(l): 111-24) and (h) isolated complementarity determining regions (CDRs).

[0127] In some embodiments, the antibodies of the present invention and their antigen-binding fragments are single-chain antibodies. In some embodiments, the present invention provides single-chain Fv (scFv), wherein the heavy chain and light chain in the Fv of the antibody of the present invention are connected into a single peptide chain by a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues). The method for producing such antibodies is described in, for example, US 4,946,778; Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore ed., Springer-Verlag, New York, pp. 269-315 (1994); Bird et al., Science, 242, 423-426 (1988); Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature, 348, 552-554 (1990). Single-chain antibodies are monovalent if only a single VH and VL are used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used.

[0128] In some embodiments, the antibodies of the present invention and antigen-binding fragments thereof are chimeric antibodies. The term "chimeric antibody" refers to a portion of a heavy chain and / or light chain that is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass and a fragment of such an antibody, as long as they exhibit desired biological activity. The present invention provides variable region antigen-binding sequences from human antibodies. Therefore, the chimeric antibodies of primary concern herein include antibodies having one or more human antigen-binding sequences (such as CDRs) and containing one or more sequences such as FR or C region sequences from non-human antibodies. In addition, chimeric antibodies as described herein are antibodies comprising a human variable region antigen-binding sequence of an antibody class or subclass and another sequence such as FR or C region sequences from another antibody class or subclass.

[0129] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are humanized antibodies. The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

[0130] In some embodiments, the antibodies of the present invention and antigen-binding fragments thereof are human antibodies or fully human antibodies. The term "human antibody" or "fully human antibody" ("humAb" or "HuMab") refers to an antibody comprising variable and constant domains derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis or during gene rearrangement or in vivo by somatic mutation).

[0131] Variant antibodies are also included within the scope of the present invention. Thus, variants of the sequences listed in the application are also included within the scope of the present invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art and are also included within the scope of the present invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can be used to improve the translation efficiency of expression systems used in antibody production.

[0132] Such variant antibody sequences have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher) sequence homology with the sequences listed in the application. Such sequence homology is calculated relative to the full length of the reference sequence (i.e., the sequence listed in the application).

[0133] The numbering of the amino acid residues in the present invention is based on Or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C., (1991), Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia, C. & Lesk, AM, (1987), Canonical structures For The Hypervariable domains Of Immunoglobulins., J. Mol. Biol., 196, 901-917. Unless otherwise specified, the numbering of amino acid residues in the present invention is based on the Kabat numbering system.

[0134] When an antibody or antigen-binding fragment thereof "specifically" binds to a region of another molecule (i.e., an epitope), it means that it reacts or binds to that epitope more frequently, more rapidly, for a longer duration, and / or with greater affinity or avidity than to another epitope. In some embodiments, an antibody or antigen-binding fragment thereof of the invention binds to or binds to that epitope with at least 10 -7 M binds to human amyloid with high affinity, especially its toxic forms, such as 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or higher. Preferably, the antibody or its antigen-binding fragment binds under physiological conditions (e.g., in vivo). Therefore, specific binding to amyloid, especially its toxic form, refers to the ability of the antibody or its antigen-binding fragment to bind to amyloid, especially its toxic form, with the above-mentioned specificity and / or under such conditions. Methods suitable for determining such binding are known in the art.

[0135] In the context of antibody binding to a specified antigen, the term "binding" generally refers to binding by an amount corresponding to about 10 -6 M or smaller K D The affinity binding, the K D The affinity of the antibody is at least 10-fold lower, such as at least 100-fold, at least 1,000-fold lower, than the affinity of the antibody for binding to a nonspecific antigen (e.g., BSA, casein) other than the designated antigen or a closely related antigen.

[0136] As used herein, the term "k d "(sec-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also called k off value.

[0137] As used herein, the term "k a "(M-1 x sec-1 or 1 / Msec) refers to the association rate constant for a specific antibody-antigen interaction.

[0138] As used herein, the term "K D "(M)" refers to the dissociation equilibrium constant for a specific antibody-antigen interaction and is expressed by k d Divide by k a get.

[0139] As used herein, the term "K A ” (M-1 or 1 / M) refers to the binding equilibrium constant for a specific antibody-antigen interaction and is expressed by k a Divide by k d get.

[0140] The antibody of the present invention is produced by any technology known in the art, such as but not limited to any chemical, biological, genetic or enzymological technology, and can be used alone or in combination. Generally, knowing the amino acid sequence of the desired sequence, those skilled in the art can easily produce the antibody by the standard techniques for producing polypeptides. For example, these antibodies can be synthesized using known solid phase methods, preferably using commercially available peptide synthesis devices (such as the device manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions to synthesize these antibodies. Alternatively, the antibody of the present invention can be synthesized by recombinant DNA technology well known in the art. For example, after the DNA sequence encoding the antibody is incorporated into an expression vector and these vectors are imported into a suitable eukaryotic or prokaryotic host for expressing the desired antibody, the antibody as a DNA expression product can be obtained, and known techniques can be used afterwards to separate the antibody from the host.

[0141] The antibodies and antigen-binding fragments thereof of the present invention can be modified by comprising any "suitable" number of modified amino acids and / or in combination with coupling substituents. In this case, "suitable" is generally determined by the ability to at least substantially retain the selectivity of amyloid proteins, especially their toxic forms, and / or the specificity of amyloid proteins, especially their toxic forms, associated with the non-derivatized parent antibody. Including one or more modified amino acids can be advantageous in, for example, increasing the serum half-life of a polypeptide, reducing polypeptide antigenicity, or increasing polypeptide storage stability. One or more amino acids can be modified, for example, simultaneously with translation or after translation during recombinant production (e.g., N-linked glycosylation on NXS / T motifs during mammalian cell expression), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprenyl (e.g., farnesylation, geranyl-geranylation) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References to amino acid modifications are commonplace in the art, see, for example, Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. The modified amino acid can, for example, be selected from a glycosylated amino acid, a pegylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid coupled to a lipid moiety, or an amino acid coupled to an organic derivatizing agent.

[0142] The antibodies and antigen-binding fragments thereof of the present invention can also be chemically modified by covalent coupling to polymers to increase their circulation half-life. Exemplary polymers and methods of attaching them to peptides are described, for example, in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., having a molecular weight of between about 1,000 and 40,000 D, such as between about 2,000 and 20,000 D, such as about 3,000 to 12,000 D PEG).

[0143] The antibodies and antigen-binding fragments thereof of the present invention can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines and insect cell lines, for example, CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (such as myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep. 18, 1-13., the contents of which are incorporated herein by reference).

[0144] The antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0145] The term "subject" refers to a warm-blooded animal, preferably a mammal (including humans, livestock and farm animals, zoo animals, sports animals or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.), more preferably humans. In one embodiment, the subject can be a "patient," that is, a warm-blooded animal, more preferably a human, who is waiting to receive or is receiving medical care or will be the subject of a medical procedure or disease progression monitoring. In one embodiment, the subject is an adult (e.g., a subject over 18 years old). In another embodiment, the subject is a child (e.g., a subject under 18 years old). In one embodiment, the subject is an elderly person who is older than 55, 60, 65, 70, 75, 80, 85, 90 years old or older. In one embodiment, the subject is a male. In another embodiment, the subject is a female.

[0146] The term "healthy subject" refers to a subject not suffering from any disease or condition or to a subject not suffering from any neurodegenerative disease.

[0147] The term "subject not suffering from Alzheimer's disease" refers to a subject who has been confirmed by formal medical procedures to not suffer from Alzheimer's disease, for example, a subject who does not suffer from mild cognitive impairment (MCI) and AD dementia as determined according to the criteria established by the National Institute on Aging and the Alzheimer's Association. In some embodiments, the subject not suffering from Alzheimer's disease is a healthy subject.

[0148] The technical solutions of the present invention will be described in detail below by way of examples, which are descriptive and illustrative and are not intended to be limiting. The reagents used in the following examples, unless otherwise noted, can be readily purchased from reagent companies such as Sigma Aldrich and Merck. The test methods, unless otherwise noted, can be found in textbooks such as Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.

[0149] Example

[0150] Example 1 Acquisition of Aβ oligomers specifically bound to 3F

[0151] 1.1 Experimental Materials and Methods

[0152] 1.1.1 Experimental Materials

[0153] Protein A magnetic beads: Bio-RAD, #1614833

[0154] Protein G magnetic beads: Bio-RAD, #1614023

[0155] Protein A / G agarose gel: Abmart, #A10001S

[0156] 6E10 antibody: Biolegend, #803002

[0157] Human Aβ42 detection kit: Immuno-Biological Laboratories

[0158] 1.1.2 Main solution

[0159] (1) 50 mM sodium hydroxide (pH 10.0);

[0160] (2) 20 mM phosphate buffered saline (PBS): pH 7.4;

[0161] (3) 0.1% PBST: PBS containing 0.1% Tween-20;

[0162] (4) 20 mM glycine eluent (pH 2.0);

[0163] (5) 1 M Tris neutralization solution: pH 10.0;

[0164] Note: The relevant reagents and solutions mentioned in Examples 2, 3, 4, 5 and 6 but without giving their sources are the same as those in Example 1.

[0165] 1.2 Preparation of mouse brain homogenate

[0166] APP / PS1 mice were deeply anesthetized with sodium pentobarbital and sacrificed after transcardial perfusion with ice-cold PBS containing heparin (10 U / mL). Brains were then removed. 1 mL of RIPA strong lysis reagent (containing protease and phosphatase inhibitors) was added to the brain tissue, and the tissue was disrupted using a Tissue Lyser II tissue grinder at 30 Hz for 8 minutes. The cells were centrifuged at 14,000 rpm for 30 minutes at 4°C, and the supernatant was collected. Prior to immunoprecipitation, the brain homogenate was added to 100 μL of Protein A / G-agarose gel and incubated at 4°C for 1 hour to remove endogenous IgG from the mouse brain homogenate. The homogenate was then added to Protein A magnetic beads cross-linked with an APP antibody to remove endogenous APP from the mouse brain.

[0167] 1.3 Antibody cross-linking with magnetic beads

[0168] First, 3F and 6E10 antibodies were cross-linked to Protein A or Protein G magnetic beads, respectively. The specific cross-linking steps were as follows:

[0169] (1) Take 200 μL of Protein A or Protein G magnetic beads and wash them three times with 2 mL of 0.1% PBST, mixing the beads thoroughly each time. Place them on a magnetic stand and discard the supernatant.

[0170] (2) Add 50 μg of 3F or 6E10 antibody to the magnetic beads, shake at room temperature for 30 min to allow the beads to fully bind to the antibody, and then wash three times with 0.1% PBST;

[0171] (3) The magnetic beads were washed once with crosslinking buffer (0.2 M triethanolamine, pH 8.2), and then 12 mg of DMP was dissolved in 2 mL of crosslinking buffer, added to the magnetic beads, and shaken at room temperature for 1 h. After the reaction, the crosslinking solution was discarded, and the beads were washed once with 2 mL of blocking buffer (0.1 M ethanolamine, pH 8.2), and then blocked with 2 mL of blocking buffer at room temperature for 2 h.

[0172] (4) After blocking, wash the magnetic beads three times with PBS, then elute them once with 0.1 M glycine (pH 2.5), react for 5 minutes, and then wash the magnetic beads three times with 0.1% PBST. Immerse the magnetic beads in PBST containing 0.02% NaN3 and store at 4°C.

[0173] 1.4 In vitro preparation of Aβo*3F

[0174] Dissolve 1 mg of Aβ42, Aβ40, or other forms of Aβ (Zhongpeptide Biochemical Co., Ltd.) in 1 mL of 100% hexafluoroisopropanol (HFIP), vortex for 5 minutes, sonicate in a water bath for 10 minutes, and then aliquot into EP tubes. Allow to evaporate overnight and store at -20°C. Prior to use, dissolve the HFIP-treated aliquot in 50 mM NaOH to a concentration of 1 mg / mL, vortex for 3-5 minutes, sonicate for 1 minute, and then dilute to 10 μM with pre-chilled PBS. Centrifuge at 21,000 g for 30-40 minutes at 4°C, and discard the precipitate (approximately 5% of the starting volume) to obtain Aβ monomers. Incubate the Aβ monomers at 25°C for 2 days and then incubate them with Protein A magnetic beads cross-linked with 3F antibody at 4°C overnight. The next day, the magnetic beads were washed three times with 0.1% PBST and eluted twice with 20-100 mM glycine (pH 2.0) for 3-5 min. The eluate was neutralized to pH 7 with 1 M Tris to obtain sAβo*3F (Aβo*3F prepared in vitro).

[0175] 1.5 Isolation and preparation of Aβo*3F from APP / PS1 mouse brain homogenate and AD patient CSF

[0176] To prepare Aβo*3F-specific Aβo (Aβo*3F), APP / PS1 mouse brain homogenate and AD patient CSF samples (obtained from the First Affiliated Hospital of Zhengzhou University, with written informed consent and approval from the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University) were incubated with Protein A magnetic beads cross-linked with 3F antibody overnight at 4°C. The next day, the beads were washed three times with 0.1% PBST and eluted twice with 20 mM-100 mM glycine (specifically, 20 mM, pH 2.0) for 3 min. The eluate was neutralized to pH 7 with 1 M Tris to obtain Aβo*3F from the brains of APP / PS1 mice (mAβo*3F, Aβo*3F isolated from the brains of AD mice) or Aβo*3F extracted from the CSF of AD patients (hAβo*3F, Aβo*3F isolated from the cerebrospinal fluid of AD patients). The mixture of Aβ aggregates after 3F immunodepletion is called Aβ-ID, which includes sAβ-ID (prepared in vitro), mAβ-ID (isolated and prepared from the brain of APP / PS1 mice), and hAβ-ID (isolated and prepared from the CSF of AD patients), and is used as a control.

[0177] Aβ aggregate mixtures, Aβ*6E10, were prepared by mixing APP / PS1 mouse brain homogenate or AD patient CSF with Protein G magnetic beads cross-linked with 6E10 antibody. The mixture was reacted overnight at 4°C and then eluted twice with 20mM-100mM glycine (specifically, 20mM, pH 2.0) for 3-5 minutes (specifically, 3 minutes). The eluate was neutralized to pH 7 with 1M Tris to obtain Aβo*6E10 from APP / PS1 mouse brain (mAβo*6E10, Aβo*6E10 isolated from AD mouse brain) or Aβo*6E10 extracted from AD patient CSF (hAβo*6E10, Aβo*6E10 isolated from AD patient CSF) (Figure 1). The concentration of immunoprecipitated Aβ was determined using an Aβ detection kit.

[0178] Example 2 Aβo*3F molecular weight and morphology characterization

[0179] 2.1 Experimental Materials and Methods

[0180] 2.1.1 Experimental Materials

[0181] Aβ peptide: mid-peptide biochemistry;

[0182] Superdex 200 10 / 300GL molecular sieve: GE Healthcare;

[0183] A11 antibody: Invitrogen, #AHB0052;

[0184] Molecular sieve protein marker: GE healthcare;

[0185] 3-8% Tris-Acetate precast gel: Invitrogen;

[0186] Carbon film supported copper mesh: Zhongke Keji;

[0187] Uranyl acetate: Zhongke Keyi;

[0188] ECL chemiluminescence kit: Pierce Biotechnology;

[0189] ThT detection reagent: Sigma-Aldrich;

[0190] Nitrocellulose membrane: Millipore.

[0191] 2.1.2 Experimental instruments

[0192] AKTA protein chromatography instrument: GE Healthcare Life Science, USA;

[0193] Protein electrophoresis instrument: Bio-rad, USA;

[0194] Protein transfer system: Bio-rad, USA;

[0195] HT7700 transmission electron microscope: Hitachi, Japan;

[0196] Safire2 TM Microplate reader: Tecan Group, Switzerland;

[0197] Amersham Imager 680 imaging system: GE, USA.

[0198] 2.1.3 Main solution

[0199] (1) 20 mM TBS buffer: Weigh 2.4 g Tris and 8 g sodium chloride, dissolve them in deionized water, adjust the pH to 7.5 with dilute hydrochloric acid, and make up to 1 L.

[0200] (2) 0.1% TBST: Add 1 mL of Tween-20 to 1 L of TBS and stir for 20 min.

[0201] (3) ThT solution: Prepare 100X ThT stock solution (500 μM) with 50 mM PB (phosphate buffer, pH = 6.5) solution and dilute to 1X with 50 mM PB (pH = 6.5) before use.

[0202] (4) 5% skim milk.

[0203] (5) Electrophoresis buffer: Dissolve 30 g Tris, 144 g Glycine, and 10 g SDS, dilute to 1 L with deionized water, and dilute 10-fold before use.

[0204] (6) 5× non-reducing loading buffer (10 mL): 2 mL 10% SDS, 0.6 mL 1 M Tris-HCl (pH 6.8), 5 mL glycerol, 1 mL 1% bromophenol blue, and dilute to 10 mL with deionized water.

[0205] (7) Transfer buffer: Dissolve 30 g Tris and 144 g Glycine in deionized water, adjust the volume to 1 L, and dilute 10-fold before use.

[0206] 2.2 Preparation and characterization of Aβ monomers, oligomers, and fibers

[0207] Preparation of Aβ monomers, oligomers, and fibers was the same as in 1.4. Aβ monomers were incubated at 25°C for 0-4 days. The aggregation state of Aβ was detected every 24 hours using ThT. The specific operation was as follows: Aβ sample was diluted to 10 μM with PBS buffer, and then 190 μL ThT detection solution and 10 μL of the sample to be tested were thoroughly mixed in a black ELISA plate and plated using Safire2 TM Fluorescence was measured using a microplate reader with an excitation wavelength of 440 nm and an emission wavelength of 480 nm. The results showed that the ThT reading of the Aβ sample gradually increased with increasing incubation time, indicating that Aβ monomers gradually aggregated into oligomers and fibrils (Figure 2, Panel A).

[0208] Dot blot experiments were used to evaluate the binding of different antibodies to Aβ monomers and aggregates. Aβ samples were spotted onto nitrocellulose membranes and blocked with 5% skim milk for 1 hour at room temperature. The membranes were incubated with different detection antibodies for 1-2 hours at room temperature, then washed three times with 0.1% TBST for 5 minutes each, and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. After three washes with 0.1% TBST for 5 minutes each, the membranes were treated with ECL chemiluminescent solution and developed using an Amersham Imager 680 imaging system. Blot size and optical density were quantified using Image J software. The results showed that 6E10 is an antibody that binds broadly to Aβ monomers, oligomers, and fibrils, with no significant difference in binding to 10 μM and 20 μM Aβ. Neither 3F nor A11 antibodies bind to Aβ monomers, and 3F and A11 antibodies bind to different oligomer types. 3F primarily binds to 10 μM Aβos, while A11 primarily binds to 20 μM Aβos, indicating that 3F and A11 recognize different oligomer conformations (Figure 2, Panel B). Furthermore, statistical analysis of the affinity of 10 μM Aβos obtained by incubation for 0-4 days with 3F showed that the affinity for 10 μM Aβos obtained by incubation for 2 days (sAβos, in vitro prepared Aβos) with 3F was the highest. Therefore, subsequent experiments used this oligomer (sAβo*3F, in vitro prepared Aβo*3F) and 3F for immunoprecipitation (Figure 2, Panel C).

[0209] 2.3 Molecular weight characterization of sAβo*3F

[0210] First, the molecular weight of sAβo*3F was detected by Western blot. The specific operation was as follows: Aβ sample was loaded onto 15% SDS-PAGE gel electrophoresis or 3-8% Tris-Acetate gel. For Tris-Acetate gel, the sample was mixed with Novex before loading. TMMix the sample with Tris-Glycine sample buffer. Electrophoresis was performed in Tris-Glycine running buffer (containing 0.5 mM DTT, 1 mM ATP, and 5 mM MgCl2) at 4°C, 150 V for 4 h. For Western blotting, proteins separated by SDS-PAGE or Tris-Acetate gel were transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk for 1-2 h at room temperature and incubated with the detection antibody (6E10) overnight at 4°C. The membrane was then washed three times with 0.1% TBST for 5 min each and incubated with the corresponding secondary antibody (HRP-conjugated goat anti-mouse secondary antibody) for 1 h at room temperature. After three washes with TBST, the membrane was covered with ECL developer and developed using an Amersham Imager 680 imaging system. Protein bands were quantified using Image J software.

[0211] The results of SDS-PAGE experiments showed that sAβo*3F was mainly composed of two oligomers with molecular weights, one of approximately 12 kDa and one greater than 180 kDa; while the native-PAGE results showed that sAβo*3F was mainly composed of one oligomer with a molecular weight of approximately 500 kDa. The difference between the two electrophoresis results may be due to the influence of SDS (Figure 2, Panel D).

[0212] To further determine the molecular weight of sAβo*3F, 500 μL of sAβo*3F or 100 μL of SEC marker were loaded onto a Superdex 200 10 / 300GL molecular sieve chromatography column connected to an AKTA pure system. The column was pre-equilibrated with Tris-Gly buffer and eluted at a flow rate of 0.5 mL / min. Comparison with the marker revealed a molecular weight of 588 kDa for sAβo*3F (Figure 2, Panels E and F).

[0213] 2.4 Molecular weight and morphology of mAβo*3F

[0214] Aβo*3F was extracted from the brains of APP / PS1 mice (mAβo*3F, Aβo*3F isolated from AD mouse brains) and its molecular weight was analyzed. Consistent with the results for sAβo*3F, mAβo*3F showed two bands on SDS-PAGE, with molecular weights of 12 kDa and >180 kDa, respectively. In native-PAGE, mAβo*3F showed a single band with a molecular weight of approximately 500 kDa (Figure 2, Panel G). SEC results showed a molecular weight of 588 kDa for mAβo*3F (Figure 2, Panel H). Furthermore, 10 μL of Aβo*3F was dropped onto a 200-mesh copper grid and adsorbed for 20 minutes, blotted dry with filter paper, and then negatively stained with 2% uranyl acetate for 30 seconds. The sample was blotted dry and air-dried. The sample was examined under a transmission electron microscope (TEM, Hitachi H7700, Japan) at a magnification of 100,000× at an operating voltage of 120 kV. The results showed that mAβo*3F was a particle with a diameter of approximately 10 nm (Figure 2, Panel H).

[0215] 2.5 Characterization of the molecular weight of Aβo*3F in CSF of AD patients

[0216] The molecular weight and distribution of Aβo*3F extracted from CSF of AD patients (hAβo*3F, Aβo*3F isolated from cerebrospinal fluid of human AD patients) were consistent with the results of sAβo*3F and mAβo*3F. hAβo*3F had only one band with a molecular weight of approximately 500 kDa in native-PAGE, while there were two bands with molecular weights of 12 kDa and greater than 180 kDa in SDS-PAGE (Figure 2, Panel I).

[0217] Example 3 Neurotoxicity Detection of Aβo*3F

[0218] 3.1 Experimental Materials

[0219] N2a cells: National Laboratory Cell Resource Sharing Service Platform (NICR);

[0220] D-polylysine hydrobromide (PDL): Sigma-Aldrich

[0221] B27 supplement (50×): Thermo Fisher Scientific

[0222] Neurobasal TM -A medium: Gibco

[0223] 70μm nylon cell strainer: BD Bioscience

[0224] 3.2 Primary neuron culture

[0225] Fetal brains of 14- to 15-day-old C57BL / 6 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were removed and the meninges were removed in HBSS (Hank's Balanced Salt Solution). The hippocampus and cortex were minced with forceps. The minced tissue fragments were transferred to a 15 mL centrifuge tube and centrifuged at 600 rpm for 3 minutes. 10 mL of 10-fold diluted trypsin digestion solution (containing DNase I) was added to the pellet and digested at 37°C for 10 minutes, gently inverting the tube every 5 minutes. After digestion, the trypsinization reaction was terminated with 40 mL of DMEM medium (containing 10% FBS), and the cell suspension was passed through a 70 μm cell sieve to remove undigested tissue fragments. Cells were harvested by centrifugation at 1200 rpm for 10 minutes, resuspended in DMEM medium, and plated into 12-well plates. After 2-4 h, the medium was changed to Neurobasal medium (containing B27, 0.5 mM L-glutamine, 0.5% penicillin and streptomycin) and cultured for 7-9 days for subsequent experiments.

[0226] 3.3 Cytotoxicity assay of sAβo*3F

[0227] This experiment primarily used the MTT assay to detect the neuronal toxicity of Aβ aggregates. The specific procedure was as follows: N2a cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. The cells were trypsinized, centrifuged, resuspended in culture medium, and seeded into a 96-well plate at approximately 5,000 cells per 100 μL of culture medium per well. After 12 hours, the cells were treated with a series of concentrations of sAβo*3F and sAβos. An equal volume of solvent was added to the cells as a control. After 72 hours, 25 μL of 5 mg / mL MTT was added to each well. After an additional 3 hours of incubation at 37°C, the medium was aspirated and 150 μL of DMSO was added. The absorbance at 570 nm and 630 nm was measured using an MD-M5 microplate reader. The average of six replicate wells was used for each sample and control, and each experiment was repeated three times. Cell viability was calculated by dividing the absorbance (background corrected) of the wells with added samples by the absorbance (background corrected) of the wells with added solvent.

[0228] MTT results showed that the half-inhibitory concentration (IC50) of sAβo*3F on cell viability was approximately 0.186 nM, while the IC50 of sAβos before immunoprecipitation was approximately 63.26 nM, a 340-fold difference in IC50 (Figure 3, Panels A and B). Furthermore, addition of 0.5 nM sAβo*3F to N2a cells caused 66% cell death, while the same concentrations of sAβos and sAβ-ID had no significant neurotoxic effect. Only when the sAβos concentration reached 200 nM did it produce 70% cytotoxicity, while at this concentration, the neurotoxicity of sAβ-ID was only 32% (Figure 3, Panel C). These results indicate that sAβo*3F is a highly neurotoxic oligomer.

[0229] 3.4 Cytotoxicity assay of mAβo*3F

[0230] MTT assays revealed that mAβo*3F exhibited an IC50 of approximately 0.111 nM for N2a cells and 0.057 nM for primary neurons (Figure 3, Panel D). Specifically, 0.5 nM mAβo*3F caused 78% cell death in primary neurons, while mAβ*6E10 and mAβ-ID exhibited no significant cytotoxicity at the same concentration. When the concentration was increased to 200 nM, mAβ*6E10 produced 54% neurotoxicity, while mAβ-ID exhibited only 20% neurotoxicity (Figure 3, Panel E).

[0231] 3.5 Cytotoxicity assay of hAβo*3F

[0232] MTT assays revealed that the IC50 of hAβo*3F against primary neurons was approximately 0.076 nM (Figure 3, Panel F). Specifically, addition of 0.2 nM hAβo*3F to primary neurons reduced cell viability by 68%, while hAβ*6E10 and hAβ-ID had no significant effect on neuronal survival at this concentration. When the concentration was increased to 40 nM, hAβ*6E10 reduced cell viability by 66%, while hAβ-ID only reduced cell viability by 40% (Figure 3, Panel G).

[0233] Example 4 Effect of Aβo*3F on Cytokine Expression Levels in Glial Cells

[0234] 4.1 Experimental Materials and Methods

[0235] 4.1.1 Experimental Materials

[0236] BV2 cells: NICR

[0237] Reverse transcription kit: Kangwei Century

[0238] UltraSYBR Mixture (Low ROX): Kangwei Century

[0239] 4.1.2 Experimental instruments

[0240] 7500Fast Real-Time PCR Instrument: Applied Biosystems

[0241] T100 Thermal Cycler PCR instrument: BIO-RAD

[0242] NanoDrop Microvolume Spectrophotometer: Quawell

[0243] 4.2 Aβo*3F can increase the expression of inflammatory factors in microglia

[0244] BV2 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells in the culture dish were trypsinized, centrifuged, and resuspended in the culture medium before being seeded in a 12-well plate with approximately 5×10^5 cells / mL of culture medium per well. After 12 hours, BV2 cells were treated with different Aβ samples, and the same volume of solvent was added to the cells as a control. After 48 hours, the expression level of intracellular proinflammatory factors was detected by fluorescence quantitative PCR (qPCR). The specific operation is as follows: Total cell mRNA was obtained by conventional method and the mRNA concentration was determined using a microtube photometer. Reverse transcription was performed according to the instructions of the reverse transcription kit to obtain cDNA, and then the expression level of the target gene was detected using EasyQuick RT MasterMix. The primers used are as follows:

[0245] mTNF-α: 5'-GATTATGGCTCAGGGTCCAA-3' (SEQ ID NO: 1),

[0246] 5'-GCTCCAGTGAATTCGGAAAG-3 (SEQ ID NO: 2);

[0247] mIL-1β: 5'-CCCAAGCAATACCCAAAGAA-3' (SEQ ID NO: 3),

[0248] 5'-GCTTGTGCTCTGCTTGTGAG-3' (SEQ ID NO: 4);

[0249] mIL-6: 5'-CCGGAGAGGAGACTTCACAG-3' (SEQ ID NO: 5),

[0250] 5'-TTGCCATTGCACAACTCTTT-3' (SEQ ID NO: 6);

[0251] GAPDH: 5'-TGAATACGGCTACAGCAACA-3' (SEQ ID NO: 7),

[0252] 5'-AGGCCCCTCCTGTTATTATG-3' (SEQ ID NO: 8).

[0253] qPCR results showed that 0.3 nM mAβo*3F, 200 nM mAβ*6E10, and 200 nM mAβ-ID induced a significant increase in the expression levels of proinflammatory cytokines in microglia, including TNF-α, IL-6, and IL-1β, but 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no obvious stimulatory effect (Figure 4A).

[0254] 4.3 Primary astrocyte culture

[0255] 1-2 day old C57BL / 6 neonatal mice were sterilized by soaking in 75% alcohol. The brains were removed and immersed in HBSS buffer. The meninges were removed and the brains were minced with tissue scissors. The pellet was placed in a 15 mL centrifuge tube and centrifuged at 600 rpm for 3 minutes. 10 mL of 10-fold diluted trypsin solution (containing DNase I) was added to the pellet and digested at 37°C for 10 minutes, gently inverting the tube every 5 minutes. After digestion, the trypsinization reaction was terminated with 40 mL of DMEM medium (containing 10% FBS), and the cell suspension was passed through a 70 μm cell sieve to remove undigested tissue fragments. Cells were harvested by centrifugation at 1200 rpm for 10 minutes, resuspended in DMEM medium, and plated into T-75 culture flasks. The cells were cultured in DMEM medium at 37°C for 5-7 days. The medium was changed every 2-3 days. After 7 days of culture, the cells were trypsinized and plated into 12-well plates for use.

[0256] 4.4 Aβo*3F can stimulate the expression of inflammatory factors in astrocytes

[0257] Primary astrocytes were treated with different Aβ samples and their effects on the expression of inflammatory factors in primary astrocytes were investigated by qPCR. The specific steps were the same as in 4.2. The primers used were as follows:

[0258] miNos: 5'-CACCTGGAACAGCACTCTCT-3' (SEQ ID NO: 9),

[0259] 5'-CTTTGTGCGAAGTGTCAGTG-3' (SEQ ID NO: 10);

[0260] mTNF-α: SEQ ID NOs: 1 and 2;

[0261] mIL-1β: SEQ ID NOs: 3 and 4;

[0262] mIL-6: SEQ ID NOs: 5 and 6;

[0263] GAPDH: SEQ ID NOs: 7 and 8.

[0264] The results showed that 0.3 nM mAβo*3F, 200 nM mAβ*6E10, and 200 nM mAβ-ID significantly increased the expression levels of proinflammatory cytokines in primary astrocytes, including TNF-α, IL-6, IL-1β, and iNos, but 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no significant effect on the expression levels of inflammatory factors in astrocytes (Figure 4, Panel B).

[0265] 4.5 Aβo*3F disrupts astrocyte-mediated synaptogenesis.

[0266] Astrocytes can induce synapse formation by secreting synaptic factors such as TSP1 and Gpc4 / 6. mAβo*3F was added to primary astrocytes, and the expression levels of various synaptic factors in astrocytes were detected by qPCR. The primers used are as follows:

[0267] mGpc4: 5'-CTGGAGGGTCCTTTCAACATT-3' (SEQ ID NO: 11),

[0268] 5'-GACATCAGTAACCAGTCGGTC-3' (SEQ ID NO: 12);

[0269] mGpc6: 5'-TAGTCCTGTATTGGCAGCCAC-3' (SEQ ID NO: 13),

[0270] 5'-GGCTAATGTCTATAGCAGGGAA-3' (SEQ ID NO: 14);

[0271] mTSP1: 5'-GGTAGCTGGAAATGTGGTGCGT-3' (SEQ ID NO: 15),

[0272] 5'-GCACCGATGTTTCTCCGTTGTGA-3' (SEQ ID NO: 16);

[0273] GAPDH: SEQ ID NOs: 7 and 8.

[0274] The results showed that treatment with 0.3 nM mAβo*3F and 200 nM mAβ*6E10 significantly reduced the expression levels of TSP1 and Gpc4 / 6, whereas treatment with 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no significant effect. Only when the concentration of mAβ-ID was increased to 200 nM did it reduce the expression of Gpc4 in astrocytes (Figure 4, Panel C).

[0275] Example 5 Effects of Aβo*3F on Mouse Cognition and Damage to Brain Neurons

[0276] 5.1 Experimental Materials and Methods

[0277] 5.1.1 Experimental Materials

[0278] Rapid Golgi staining kit: FD NeuroTechnologies

[0279] Nissl staining kit: Solebro

[0280] 5.1.2 Experimental instruments

[0281] Brain stereotactic injection system: Chinese Academy of Medical Sciences

[0282] Y-maze equipment: Chinese Academy of Medical Sciences

[0283] New object identification equipment: Chinese Academy of Medical Sciences

[0284] 5.2 Experimental Animals

[0285] Three-month-old C57BL / 6 mice were used. All mice were housed in a cleanroom at 22 ± 2°C and 45% ± 10% humidity with free access to food and water. The mice were maintained on a 12-h light / 12-h dark cycle. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals in Public Health Services of China. Experiments involving mice were approved by the Institutional Animal Care and Use Committee of Tsinghua University.

[0286] 5.3 Stereotaxic Brain Injection

[0287] Three-month-old C57BL / 6 mice were randomly divided into six groups, with 6-8 mice in each group, namely: a group injected with 2.5 nM (45.5 pg) mAβo*3F, a group injected with 2.5 nM (45.5 pg) mAβ*6E10, a group injected with 2.5 nM (45.5 pg) mAβ-ID, a group injected with 600 nM (10.9 ng) mAβ*6E10, a group injected with 600 nM (10.9 ng) mAβ-ID, and a control group injected with Tris-Gly solvent. Mice were deeply anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg) and secured to a stereotaxic injection station. The skin of the mouse brain was incised along the midline to expose the skull. The injection site was located using a stereotaxic instrument: bregma as the starting point, AP = -1.7 mm, ML = ±1.0 mm, DV = -1.5 mm. A craniotomy was performed using a cranial drill along the designated point, and bilateral injections were performed, with 4 μL injected into each hemisphere at an injection rate of 0.4 mL / min. After the injection, the needle was left in place for 3 minutes to allow complete absorption of the sample. The surgical site was cleaned with sterile saline, and the incision was sutured. Postoperatively, the mice were monitored and provided with postoperative care. Behavioral and cognitive abilities of the mice were assessed 24 hours after the stereotaxic injection, and the mice were then dissected for physiological and biochemical analyses.

[0288] 5.4 Aβo*3F severely impairs the memory ability of mice

[0289] The novel object recognition test and Y-maze test were used to detect changes in the cognitive abilities of mice.

[0290] 5.4.1 Novel Object Recognition Experiment

[0291] The novel object recognition experiment is designed based on the spontaneous tendency of mice to show more interaction with new and unfamiliar objects. The experiment is mainly divided into three stages:

[0292] (1) Familiarization phase: Prepare a white box (40 cm wide, 40 cm deep, and 40 cm high). Allow each mouse to freely explore the box for 5 min without any objects inside. 1 h later, inject the mouse with Aβ samples.

[0293] (2) Training phase: 24 hours later, two identical objects were placed in the box. Each mouse was placed in the same box as in the first phase and allowed to explore freely for 5 minutes. The number of times the mouse sniffed and touched each object was recorded.

[0294] (3) Testing phase: After 6 h of training, the right object was replaced with a new object of different material, color, and shape. The mice were allowed to explore freely for another 5 min. The number of times the mice sniffed and touched the two objects was recorded to test their memory ability. The discrimination index was determined using the following formula: (number of times the new object was touched – number of times the old object was touched) / (number of times the new object was touched + number of times the old object was touched).

[0295] The results showed that compared with the control group, the discrimination index of mice treated with 2.5nM mAβo*3F and 600nM mAβ*6E10 for new objects was significantly decreased, while the discrimination index of mice in other Aβ-treated groups for new objects was not significantly different from that of the control group, indicating that Aβo*3F significantly reduced the memory ability of mice (Figure 5A).

[0296] 5.4.2 Y-maze experiment

[0297] After the novel object recognition experiment, the mice's spatial memory ability was tested using a Y-maze experiment. The Y-maze consists of three identical arms, each with a 120-degree angle between them. Each arm measures 8cm x 30cm x 15cm (width x length x height). The experiment was divided into two phases:

[0298] (1) During the training phase, the three arms were randomly named A, B, and C. Arms A and B were open throughout the experimental phase, while arm C, as a novel arm, was closed during the training phase. After being placed in arm A, each mouse was allowed to freely explore arms A and B for 10 min.

[0299] (2) After 1 hour of training, the C arm was opened and the mice were placed in the A arm again in order. The mice were allowed to explore the A, B, and C arms for 5 minutes, and the time they spent in the three arms was analyzed. All experimental processes were recorded by a camera installed above the Y maze.

[0300] The results showed that the duration of time spent in the novel arm by mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group. However, mice injected with 2.5 nM mAβ*6E10, 2.5 nM mAβ*ID, and 600 nM mAβ*ID showed no significant difference from the control group. This suggests that mAβo*3F, but not mAβ*6E10 or mAβ-ID, causes severe memory impairment in mice at low concentrations. For mAβ*6E10, only when the concentration is increased to 600 nM does a similar degree of memory impairment occur in mice (Figure 5, Panel B).

[0301] 5.5 Aβo*3F significantly reduced the dendritic spine density of mouse neurons

[0302] Mouse brain samples were stained using the FD Fast Golgi Staining Kit. 24 hours before dissection, solutions A and B in the kit were mixed at a 1:1 ratio and stored in the dark. After sacrifice, the mice were harvested without perfusion and the brains were separated along the midline into left and right hemispheres. The left hemisphere was rinsed with PBS to remove any surface blood. The surface solution was blotted dry with filter paper, and the tissue was then placed in the pre-prepared solution A+B. After 24 hours, the solution was changed and the tissue was stored at room temperature in the dark for 2 weeks. During this soaking period, the tissue was gently inverted twice weekly for optimal results. After 2 weeks, the tissue was transferred to solution C, and the solution was changed after 24 hours. The tissue was stored at room temperature in the dark for 5 days. 100 μm thick cryosections were then obtained using a freezing microtome at -22°C. After the sections were allowed to air dry at room temperature, staining was initiated. The sections were first washed twice with Milli-Q water for 4 minutes each. Then, the working staining solution was prepared and the sections were stained in the working staining solution for 15 minutes. The sections were then washed twice with Milli-Q water for 5 minutes each. After staining, sections were dehydrated with 50%, 75%, 95%, and 100% ethanol, with each concentration gradient dehydration lasting 4 minutes. Sections were then placed in xylene and mounted with neutral resin for observation. Dendrites and spines of neurons in the CA1 region of the hippocampus were observed using an Olympus inverted microscope with a 100× lens objective. Spine density was determined using Image J software (dendritic density = number of spines / dendrite length).

[0303] By calculating the dendritic spine density, it was found that compared with the control group, the dendritic spine density of neurons in the CA1 region of the mouse brain treated with 2.5nM mAβo*3F and 600nM mAβ*6E10 was significantly decreased, but 2.5nM mAβ*6E10, 2.5nM mAβ-ID or 600nM mAβ-ID had no significant effect on the dendritic spine density of mouse brain neurons (Figure 5C, D).

[0304] 5.5 Aβo*3F significantly reduces the number of neurons in the mouse hippocampus

[0305] 5.5.1 Paraffin embedding and sectioning

[0306] After the behavioral experiment, mice were deeply anesthetized with sodium pentobarbital (50 mg / kg) and perfused transcardially with ice-cold PBS containing heparin (10 U / mL). Finally, they were sacrificed and dissected. The brains of the mice were removed and divided into the left and right hemispheres along the midline. The left hemisphere was fixed in 4% paraformaldehyde for 48 hours and then dehydrated in a gradient of ethanol: 50%, 70%, 80%, and 90% ethanol for 1 hour each. This was repeated with 100% ethanol for 1 hour, followed by 50% xylene for 30 minutes. After dehydration, the tissues were immersed in paraffin for 4 hours and embedded. 5 μm tissue sections were obtained using a paraffin tissue slicer.

[0307] 5.5.1 Nissl staining

[0308] 5μm paraffin sections were heated at 60°C for 30 minutes and then dewaxed by immersion in 100% xylene, 50% xylene, and 100% ethanol for 10 minutes each, and then in 75% ethanol, 50% ethanol, 30% ethanol, and deionized water for 5 minutes each. Following the Nissl staining instructions, cresyl violet solution was added dropwise to the sections, heated at 56°C for 1 hour, and allowed to incubate at room temperature for 1-3 minutes until the background was nearly colorless. The sections were then destained with graded alcohols (70%, 95%, and 100%; 20 seconds each), immersed in xylene, and mounted with neutral resin. Data were collected and analyzed using a microscope. The results showed that 2.5 nM mAβo*3F and 600 nM mAβ*6E10 caused a significant decrease in the number of neurons in the CA1 and DG regions of the mouse brain, while 2.5 nM mAβ*6E10, 2.5 nM mAβ-ID, or 600 nM mAβ-ID had no significant effect on the number of neurons (Figure 5, E, F, and G).

[0309] Example 6 Aβo*3F activates glial cells in the mouse brain to produce neuroinflammation

[0310] 6.1 Experimental Materials and Methods

[0311] 6.1.1 Experimental Materials

[0312] Anti-GFAP antibody: Cell Signaling Technology, #3670

[0313] Anti-Iba-1 antibody: GeneTex, #GTX101495

[0314] BCA kit: Thermo Fisher Scientific

[0315] RIPA strong lysis buffer Solebo

[0316] Protease inhibitors: Millipore

[0317] Phosphatase inhibitor (100×): Solebol

[0318] Mouse IL-1β detection kit: Biolegend

[0319] Mouse IL-6 detection kit: Biolegend

[0320] 6.1.2 Experimental instruments

[0321] MD-M5 microplate reader: Molecular Devices, USA

[0322] Tissue LyserⅡ tissue grinder: Qiagen

[0323] 6.1.3 Immunohistochemistry

[0324] Deparaffinized 5 μm paraffin sections were immersed in citric acid antigen retrieval solution (3 g trisodium citrate, 0.4 g citric acid dissolved in deionized water, diluted to 1 L), boiled in a water bath for 15 minutes, and allowed to cool naturally at room temperature. After antigen retrieval, sections were washed three times with PBS for 5 minutes each, then fixed with 80% (vol / vol) methanol containing 0.3% H₂O₂ to remove endogenous catalase. Sections were then washed three times with PBS for 5 minutes each, permeabilized in 0.3% Triton X-100 for 20 minutes at room temperature, and washed three times with PBS for 5 minutes each. Sections were blocked with 10% donkey serum for 1 hour at room temperature and incubated with the corresponding primary antibody overnight at 4°C. The next day, sections were washed three times with PBS for 5 minutes each, incubated with the corresponding secondary antibody for 1 hour at room temperature, washed three times with PBS, and visualized.

[0325] 6.2 Aβo*3F can activate microglia and astrocytes

[0326] Staining for the hippocampal microglial marker Iba-1 revealed that 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly activated microglia in the DG and CA1 regions of the hippocampus and increased Iba-1 expression in these cells. However, treatment with 2.5 nM mAβ*6E10 or mAβ-ID had no significant effect (Figure 6, Panels A, C, and D). Furthermore, 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly induced DG microglia to enter an amoeboid state with enlarged cell bodies and fewer branches, representing a hyperactivated microglia state (Figure 6, Panel A). By staining the astrocyte marker GFAP in the mouse hippocampus, it was found that injection of 2.5 nM mAβo*3F and 600 nM mAβ*6E10 into the mouse brain activated astrocytes in the DG and CA1 regions of the hippocampus and increased the expression level of GFAP in the hippocampus (Figure 6B, E, and F).

[0327] 6.3 Aβo*3F increases the levels of inflammatory factors in the hippocampus of mice

[0328] IL-6 and IL-1β ELISA kits were used to measure inflammatory cytokine levels (IL-6 and IL-1β) in the mouse brain, respectively, according to the manufacturer's instructions. Briefly, brain homogenate was diluted to a certain multiple and added to an ELISA plate pre-coated with the corresponding capture antibody. The corresponding detection antibody and secondary antibody were then added. The absorbance was measured at 450 nm using a MD-M5 microplate reader with TMB as the substrate. The results showed that injection of 2.5 nM mAβo*3F, 600 nM mAβ*6E10, and 600 nM mAβ-ID significantly increased IL-1β and IL-6 levels in the hippocampus of mice. However, IL-1β and IL-6 levels in the brains of mice injected with 2.5 nM mAβ*6E10 or 2.5 nM mAβ-ID were not significantly different from those in the control group (Figure 6, Panels G and H).

[0329] Example 7 Detection of Aβo*3F Levels in CSF and Plasma of AD Patients (MSD Method)

[0330] 7.1 Experimental Materials and Methods

[0331] 7.1.1 Experimental Materials

[0332] Aβ Multifactor Detection Kit (4G8): Meso Scale Diagnostics

[0333] 7.1.2 Experimental instruments

[0334] MSD-S600 electrochemiluminescence detector Meso Scale Diagnostics, USA

[0335] 7.2 Detection of Aβo*3F Levels in Plasma and CSF of AD Patients

[0336] To evaluate the relevance of Aβos specifically recognized by 3F (Aβo*3F) and the pathogenesis of AD patients, the levels of Aβ42 and Aβ40 contained in Aβo*3F isolated from CSF and plasma of AD patients were tested.

[0337] The use of human plasma and CSF samples in this study was approved by the Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University, and all subjects provided written informed consent before participating in the study. Basic information about the subject samples used in this study is as follows: plasma samples were collected from 20 patients with mild cognitive impairment (MCI) with a mean age of 73.3 years (range, 57–84 years), 20 patients with AD with a mean age of 69.4 years (range, 52–85 years), and 20 healthy individuals with a mean age of 70.9 years (range, 65–78 years). In addition, CSF samples were collected from seven AD patients with a mean age of 68 years (range, 59–77 years) and seven non-demented subjects with a mean age of 66.3 years (range, 48–79 years).

[0338] To extract Aβo*3F from the plasma and CSF of AD patients and healthy elderly individuals, human plasma or CSF samples were first added to 100 μL of Protein A / G-Sepharose and incubated at 4°C for 1 hour to remove endogenous IgG. The samples were then incubated with Protein A magnetic beads cross-linked with 3F antibody for 2 hours at room temperature. The next day, the beads were washed three times with 0.1% PBST and eluted twice with 20 mM glycine (pH 2.0) for 3 minutes. The eluate was neutralized to pH 7 with 1 M Tris. The concentration of Aβo*3F extracted by immunoprecipitation was measured using the MSD Aβ Multifactor Assay Kit (4G8). The results showed that the levels of Aβ42 and Aβ40 contained in the Aβo*3F in the CSF and plasma of AD patients were significantly increased compared to those in healthy elderly individuals (Figure 7). Furthermore, the levels of Aβ42 and Aβ40, a component of Aβo*3F, in the plasma of MCI patients were significantly higher than those in controls, and both levels increased gradually with the progression of AD pathology (Figure 7, Panels C and D). These results indicate that Aβo*3F levels in the CSF and plasma of patients with AD and AD-induced MCI are significantly higher than those in healthy elderly individuals and are closely correlated with the progression of AD pathology, suggesting that Aβo*3F may be a biomarker for the diagnosis of AD, particularly in the early stages of AD. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test in GraphPad Prism 8 software. 95% confidence intervals are given. * indicates P < 0.05, indicating a statistically significant difference; ** indicates P < 0.01, indicating a very significant difference; *** indicates P < 0.001, indicating a very significant difference; and **** indicates P < 0.0001, indicating a very significant difference.

[0339] Example 8 Detection of Aβo*3F Levels in Plasma of AD and MCI Patients (Chemiluminescence Method)

[0340] 8.1 Experimental Materials and Methods

[0341] 8.1.1 Experimental Materials

[0342] 102 Antibody: Beijing Regeneron Biotechnology Co., Ltd.

[0343] 8.1.2 Experimental instruments

[0344] MG60 pro chemiluminescence detector: Beijing Rejing Biotechnology Co., Ltd., Beijing

[0345] 8.2 Detection of Aβo*3F Levels in Plasma of AD Patients

[0346] To evaluate the relevance of Aβos specifically recognized by 3F (Aβo*3F) and the pathogenesis of AD patients, the levels of Aβ42 contained in Aβo*3F isolated from the plasma of AD patients were tested.

[0347] The human plasma and CSF samples used in this study were approved by the Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University, and all subjects provided written informed consent before participating in the study. The basic information of the subject samples used in this study is as follows: plasma samples were collected from three patients with AD (mean age 65.6 years, range 62-75 years), three patients with MCI (mean age 63.3 years, range 62-65 years), and three healthy individuals (mean age 63.6 years, range 61-65 years).

[0348] To extract Aβo*3F from the plasma of AD patients and healthy elderly individuals, human plasma samples were first added to 200 μL of Protein A-Sepharose and incubated at room temperature for 15 minutes to remove endogenous IgG. The samples were then incubated with JSR magnetic beads coated with 3F antibody for 2 hours at room temperature. The next day, the beads were washed three times with 0.1% PBST and eluted twice with 20 mM glycine (pH 2.2) for 5 minutes. The eluate was neutralized to pH 7 with 1 M Tris. The concentration of Aβo*3F extracted by immunoprecipitation was measured using the 3F-102 detection kit. The results showed that the levels of Aβ42 contained in Aβo*3F in the plasma of AD patients were significantly increased compared to those in healthy elderly individuals (Figure 8). In addition, the levels of Aβ42 contained in Aβo*3F in the plasma of patients with MCI were significantly higher than those in the control group (Figure 8). These results indicate that the plasma levels of Aβo*3F in AD patients and MCI patients are significantly higher than those in healthy elderly people, indicating that Aβo*3F can be used as a biomarker for AD diagnosis, especially for early diagnosis of AD.

[0349] Example 9: Diagnosis of AD by detecting Aβo*3F levels in CSF or plasma

[0350] The human plasma and CSF samples from Example 7 were shuffled and renumbered, then given to blinded researchers for enrichment of Aβo*3F using Protein A magnetic beads cross-linked with the 3F antibody according to the method of Example 7. The levels of Aβ42 and Aβ40 contained in the Aβo*3F samples were then measured using the MSD Aβ Multifactor Detection Kit (4G8). The results showed that the amounts of Aβ42 and Aβ40 contained in the Aβo*3F samples were positively correlated with AD pathology. This method for measuring Aβo*3F levels in human plasma and CSF samples was able to significantly distinguish patients with AD and MCI from healthy controls (Figure 9).

[0351] Example 10 Optimization of Antibodies for Detecting Aβo*3F Oligomers

[0352] The 3F antibody was mutated from amino acid K at position 100 to amino acid R (not counting the MA residue introduced for single-chain antibody expression, representing position 98 in the antibody numbering system; this variant is designated K98R mutant, with the sequence shown in SEQ ID NO: 24). Binding of the K98R mutant to Aβo*3F oligomers was studied using the following method.

[0353] The above Aβ oligomers were coated onto 96-well plates at 100 ng / well and coated overnight at 4°C. The next day, the plates were blocked with 3% BSA at room temperature for 2 hours, washed twice with PBST, and patted dry for later use. K98R mutant and 3F were diluted, starting from 1 μg / ml, and serially diluted for a total of 14 gradients. The diluted antibody solution was added to the ELISA plate coated with the above Aβ oligomers, and parallel wells were made. The plates were incubated at 37°C for 1 hour, washed three times with PBST, and patted dry. 1:10000 diluted goat anti-human IgG-HRP was added, and the plates were incubated at 37°C for 45 minutes, washed three times with PBST, and patted dry. TMB color development solution was added for about 15 minutes. After the stop solution was stopped, the OD 450 The readings were taken in nm, and the values ​​were analyzed, compared and plotted using GraphPad.

[0354] The results are shown in Figure 10. The experiment shows that the binding affinity of the K98R mutant to Aβo*3F oligomers is further improved, suggesting that the higher binding affinity of this mutant to Aβo*3F oligomers will help to more sensitively detect the presence of Aβo*3F oligomers in the subject, thereby diagnosing whether the subject has a neurodegenerative disease at an earlier stage. The present inventors considered the reasons for this result and discovered that, when numbered according to IMGT numbering, amino acid position 98 is also the residue of the heavy chain CDR3 of the 3F antibody (according to IMGT numbering, the heavy and light chain CDR sequences of the 3F antibody are: CDR-H1: GFTFSSYA (SEQ ID NO: 25); CDR-H2: ISNLGLTT (SEQ ID NO: 26); CDR-H3: AKTTSRFDY (SEQ ID NO: 27); CDR-L1: QSISSY (SEQ ID NO: 28); CDR-L2: KAS; CDR-L3: QNSAVRPVT (SEQ ID NO: 29)). The present inventors also mutated several other variant sequences with the K98R mutation, with similar results (data not shown).

[0355] Equivalent plan

[0356] Although multiple embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily envision various other means and / or structures for achieving the functions described herein and / or obtaining the results and / or one or more advantages described herein, and will consider that each such variation and / or modification is within the scope of the present invention. More broadly, a person skilled in the art will readily understand that all parameters, materials, and settings described herein are intended to be exemplary, and that actual parameters, materials, and / or settings will depend on the specific application in which the teachings of the present invention are used. A person skilled in the art will recognize or be able to determine many equivalents to the specific embodiments of the present invention described herein using only routine experimentation. Therefore, it should be understood that the foregoing embodiments and examples are presented by way of example only, and within the scope of the appended claims and their equivalents, the present invention may be implemented in a manner different from that specifically described and claimed. Any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention if such features, systems, articles, materials, and / or methods do not conflict with each other.

[0357] As used in the present specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present in conjunction in some cases and not in other cases. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated otherwise. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a reference to "A and / or B" may, in one embodiment, mean A but not B (optionally including other elements in addition to B); in another embodiment, mean B but not A (optionally including elements in addition to A); in yet another embodiment, mean both A and B (optionally including other elements); and so on.

[0358] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be understood to be inclusive, i.e., including at least one of a plurality of elements or a list of elements, but also including more than one, and optionally, other unlisted items. Only when a contrary term is explicitly indicated, such as "only one of" or "exactly one of", or when used in a claim, "consisting of..." will refer to exactly one element of a plurality of elements or a list of elements. Generally, when preceded by an exclusive term, such as "either", "one of", "only one of" or "exactly one of", the term "or" as used herein should only be understood to represent an exclusive alternative (i.e., "one or the other but not both"). "Substantially consisting of...", when used in a claim, should have its ordinary meaning in the field of patent law.

[0359] As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements of the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") may mean at least one, optionally including more than one A, with no B present (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one B, with no A present (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0360] In the claims and the foregoing description, all conjunctions such as "comprises," "includes," "with," "having," "contains," "involving," "possessing," etc. are to be understood as open-ended, i.e., meaning including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively.

[0361] The use of ordinal terms in the claims, such as "first," "second," "third," etc. to modify claim elements does not in itself imply any priority, precedence, or order of one claim element relative to another claim element, or the temporal order in which actions in a method are performed, but merely serves as a label to distinguish one claim element having a certain name from another element having the same name (but for ordinal terms) to distinguish claim elements.

Claims

1. A method for diagnosing whether a subject has early-stage or mid-stage Alzheimer's disease (AD) or mild cognitive impairment (MCI) due to AD, or is at risk of having AD, the method comprising the steps of: a) optionally, providing a sample to be tested from a subject, b) contacting a sample from the subject with a reagent for detecting the presence and / or level of Aβo*3F in the sample from the subject, c) detecting the presence and / or level of Aβo*3F in a sample from the subject, in, The presence and / or level of Aβo*3F in the subject sample indicates that the subject suffers from AD or AD-induced MCI, wherein the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody, and based on size exclusion chromatography analysis, its molecular weight is approximately 588 kDa, and the light and heavy chain CDR sequences of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively. 2 . The method according to claim 1 , wherein the subject is a patient suspected of having AD or AD-induced MCI, preferably, the subject is a human, a non-human primate, a cat or a dog. 3 . The method according to claim 1 , further comprising clinical neuropsychological and neuroimaging assessment steps, preferably, the neuroimaging is Aβ-PET scanning imaging and / or tau-PET.

4. The method according to any one of claims 1 to 3, further comprising detecting changes in tau levels in the subject sample, preferably, the tau level is the total tau level or the phosphorylated tau level.

5. A kit for diagnosing whether a subject has AD or AD-induced MCI or is at risk of developing AD, comprising a reagent for detecting the presence and / or level of Aβo*3F in a sample from the subject, wherein the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody, and has a molecular weight of approximately 588 kDa based on size exclusion chromatography analysis. The light and heavy chain CDR sequences of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively.

6. The method according to any one of claims 1 to 4 or the kit according to claim 5, which relates to or includes an enrichment reagent for Aβo*3F, wherein the enrichment reagent for Aβo*3F includes a binding agent that specifically binds to Aβo*3F. Preferably, the binding agent that specifically binds to Aβo*3F is an antibody that specifically binds to Aβo*3F. Preferably, the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof. More preferably, the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, diabody or bispecific antibody.

7. The method or kit according to any one of claims 1 to 6, wherein the reagent for detecting the presence and / or level of Aβo*3F comprises a first binding agent that specifically binds to Aβo*3F or Aβ aggregates, preferably, the first binding agent that specifically binds to Aβo*3F or Aβ aggregates is an antibody that specifically binds to Aβo*3F or Aβ aggregates, preferably, the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof, more preferably, the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, diabody or bispecific antibody.

8. The method or kit of claim 7, wherein the first binding agent is linked to a detection agent that allows its detection.

9. The method or kit according to claim 7, wherein the reagent for detecting the presence and / or level of Aβo*3F further comprises a second binding agent, which specifically binds to the first binding agent. Preferably, the second binding agent is an antibody that specifically binds to the first binding agent. Preferably, the antibody that specifically binds to the first binding agent is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof. More preferably, the antigen-binding fragment is selected from scFv, F(ab')2, Fab2, Fab, Fab', Fv, Fd, dAb, camel antibody, nanobody, diabody or bispecific antibody. Preferably, the antibody that specifically binds to the first binding agent is linked to a detection agent that allows its detection.

10. The method or kit according to claim 8 or 9, wherein the detection agent is selected from the group consisting of chemiluminescent labels, electrochemiluminescent labels, chromophores, fluorescent labels, fluorescein-type labels, umbelliferone, lissamine, cyanine, Texas Red, BODIPY (Invitrogen) or its analogs, paramagnetic labels, radiolabels, biotin, streptavidin / biotin, avidin / biotin, haptens, digoxigenin, metal complexes, metals, enzymes, colloidal gold, or combinations thereof.

11. The method or kit according to any one of claims 1 to 10, wherein the detection is selected from the group consisting of chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology (Simoa), flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot, protein chip, positron emission tomography and / or single photon emission computed tomography. Preferably, the kit comprises reagents, materials, containers and / or devices required for performing the detection selected from the group consisting of chemiluminescence assay, electrochemiluminescence assay, enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunochromatography assay, radioimmunoassay, single molecule immunoassay technology, flow cytometry, cell sorting, immunoprecipitation assay, immunodiffusion assay, dot blot assay, Western blot and / or protein chip. Preferably, the enzyme-linked immunosorbent assay is selected from the group consisting of direct enzyme-linked immunosorbent assay, indirect enzyme-linked immunosorbent assay, direct sandwich enzyme-linked immunosorbent assay and indirect sandwich enzyme-linked immunosorbent assay.

12. The method or kit according to any one of claims 1 to 11, wherein the subject sample is selected from cells, tissues, organs and / or body fluids of the subject, preferably, the body fluid is selected from whole blood, plasma, serum, cerebrospinal fluid, lymph, saliva, synovial fluid, bronchoalveolar lavage fluid, sputum, ascites, urine, amniotic fluid, peritoneal fluid, pericardial fluid, semen and / or vaginal secretions, preferably, the body fluid is selected from whole blood, plasma, serum and / or cerebrospinal fluid.

13. The method or kit according to any one of claims 7 to 12, wherein the first binding agent that specifically binds to Aβo*3F is attached to a solid support.

14. The method or kit according to any one of claims 1 to 13, wherein the method further comprises a step of detecting a control sample, and / or the kit further comprises a control sample, wherein the control sample is from a healthy subject or a subject not suffering from AD.

15. The method or kit according to any one of claims 6 to 14, wherein the antibody that specifically binds to Aβo*3F is a polyclonal antibody and / or monoclonal antibody or an antigen-binding fragment thereof obtained by immunization against Aβo*3F. Preferably, the antibody is a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a sheep antibody or a non-human primate antibody.

16. The method or kit according to any one of claims 1 to 15, wherein Based on the MSD electrochemiluminescence method, the concentrations of Aβo*3F in the cerebrospinal fluid of healthy people were Aβ42o*3F: 80.44±20.88pg / ml, Aβ40o*3F: 24.35±5.08pg / ml; and / or the concentrations of Aβo*3F in plasma were Aβ42o*3F: 71.63±36.8pg / ml, Aβ40o*3F: 2.24±0.92pg / ml; the concentrations of Aβo*3F in the plasma of people with mild cognitive impairment were Aβ42o*3F: 1 The concentrations of Aβo*3F in the cerebrospinal fluid of AD patients were Aβ42o*3F: 264.8±42.26 pg / ml, Aβ40o*3F: 85.74±10.62 pg / ml, and / or the concentrations of Aβo*3F in plasma were Aβ42o*3F: 159.44±36.8 pg / ml, Aβ40o*3F: 14.0±5.59 pg / ml; or Based on the chemiluminescence method, the concentration of Aβ42o*3F in the plasma of healthy people was 68.02±39.17pg / ml; the concentration of Aβ42o*3F in the plasma of people with mild cognitive impairment was 124.5±12.57pg / ml; and the concentration of Aβ42o*3F in the plasma of AD patients was 205.75±50.96pg / ml.

17. The method or kit according to any one of claims 1 to 16, wherein Based on the MSD electrochemiluminescence method and the chemiluminescence method, the sensitivity of the detection is as low as 0.5 pg / ml.

18. The method or kit according to any one of claims 7 to 17, wherein the antibody that specifically binds to Aβo*3F is a plurality of antibodies specific for different epitopes of Aβo*3F, such as two, three, four, five or more antibodies that are respectively specific for two, three, four, five or more different epitopes of Aβo*3F.

19. The method or kit according to claim 6, wherein the step of enriching Aβo*3F from the sample is performed by immunoprecipitation using an antibody that specifically binds to Aβo*3F.

Images