Highly toxic amyloid protein oligomers and their uses

Abstract

The present invention provides a novel, highly toxic amyloid protein oligomer, Aβo*3F, which specifically binds to 3F antibodies and is the most predominant toxic component in the Aβ oligomer mixture, with potent pathogenic effects. The present invention also provides a binding agent that specifically binds to Aβo*3F, a composition comprising Aβo*3F, and the use of Aβo*3F as a target for preventing, treating, and / or diagnosing MCI and / or AD in subjects.

Description

[Technical Field]

[0001] The present invention relates to a novel highly toxic amyloid protein oligomer and uses thereof. More specifically, the present invention relates to a newly isolated highly toxic amyloid protein oligomer, Aβo*3F, a method for producing the same, an immunogenic composition containing the same, a vaccine, an antibody against the same, and uses thereof. [Background technology]

[0002] Alzheimer's disease (AD, commonly known as senile dementia) is a chronic neurodegenerative disease. Approximately 50 million people worldwide currently suffer from AD. To date, there are no specific and effective treatments or treatments available, which has already placed a heavy burden on humanity. AD progression is relatively slow. Initially, patients exhibit no obvious clinical symptoms, but gradually progress to memory loss and personality and behavioral changes. In the later stages, AD patients experience widespread neuronal death, significant cerebral atrophy, and treatment becomes less effective. Recent clinical trials have shown that early detection and early intervention in AD patients can slow the progression of AD. However, to achieve early treatment and intervention for AD patients, timely and accurate differential diagnosis and diagnosis are essential. However, ideal early diagnostic methods and technologies currently in clinical use are lacking. Research has shown that changes in amyloid beta protein (Aβ), which trigger the onset and progression of AD, appear 15 to 20 years before clinical symptoms appear.

[0003] The pathological hallmarks of AD are the formation of senile plaques, which are aggregates of Aβ, and neurofibrillary tangles, which are aggregates of tau protein, in the brain. Aβ can aggregate into oligomers, protofibrils, and mature fibrils. Aβ oligomers are the most neurotoxic aggregate form, and many forms, such as Aβ dimers, trimers, and amyloid beta-derived diffusible ligands (ADDLs), have been reported. Aβ oligomers are divided into two types, low molecular weight and high molecular weight, based on molecular weight, with high molecular weight oligomers exhibiting greater neurotoxicity. Aβ dimers are the smallest Aβ oligomers and are generally believed to be the basic building block of Aβ oligomers. Aβ dimers are elevated in the brains of AD patients and AD transgenic mice, are stable to SDS and strong denaturing agents, and exhibit some degree of neurotoxicity. In addition to Aβ dimers, Aβ trimers are also thought to be the aggregation unit of multiple 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 unclear. Furthermore, Aβ oligomers can further aggregate to form spherical oligomers (amylospheroids, ASPDs) with a diameter of approximately 12 nm and diffusible oligomers (ADDLs) with a diameter of 5–6 nm. Both of these polymer forms are thought to be Aβ oligomers with unique conformations and neurotoxicity. Although multiple Aβ oligomer forms have been identified, it is still unknown which specific type or types of Aβ oligomers are most toxic and play a key role in the development and progression of AD. This may explain the limited success of drug development against Aβ oligomers. Furthermore, the instability of Aβ oligomers limits their extraction and study to a single conformation. Therefore, improving Aβ oligomer extraction and detection techniques is one of the key points to clarify the types of Aβ oligomers and their corresponding functions.Furthermore, 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 of Aβ oligomers and the development of therapeutic agents.

[0004] A large body of research has shown that the severity of neurodegenerative diseases is closely related to the levels of amyloid protein oligomers in patients' brains. Aβ oligomers play a key role in the development and progression of AD by causing functional neuron death, cognitive impairment, and dementia. However, there are numerous types of Aβ oligomers, which can affect central nervous system function through multiple mechanisms of action. Various Aβ oligomers vary in toxicity, with significant differences in size, conformation, aggregation morphology, toxicity, and time to brain appearance. While numerous studies have shown that some Aβ oligomers, such as dimers, trimers, and ADDLs, can exert neurotoxic effects, our understanding of the Aβ oligomer forms that exert truly significant pathogenic effects remains limited. Therefore, research into Aβ oligomer toxicity should focus on identifying the types of Aβ oligomers and their corresponding toxic mechanisms, rather than focusing on oligomer mixtures. Identifying key toxic oligomers closely associated with the development and progression of AD could provide ideal markers for early warning and diagnosis of AD, as well as pinpointing ideal targets for AD treatment.

[0005] Immunotherapy has long been the focus of research in the field of AD treatment, and Aβ antibodies and vaccines have undergone the development of three generations of products targeting the entire Aβ molecule, the N-terminus of Aβ, and the spatial structure of Aβ aggregates. In recent years, dozens of Aβ-targeting antibodies and vaccines have entered the clinical trial stage. These preparations have shown good therapeutic effects in animal trials, significantly improving the cognitive level of AD transgenic animals and reducing the amount of senile plaques and other pathological changes in the animal's brain. However, due to poor therapeutic effects or the occurrence of serious side effects in AD clinical trials, they have not yet been able to undergo Phase III clinical trials. In June 2021, the U.S. Food and Drug Administration (FDA) approved the launch of Aduhelm (aducanumab, a monoclonal antibody) through its accelerated approval program. This antibody can specifically recognize Aβ oligomers and has shown some therapeutic efficacy in clinical trials. However, this antibody has been associated with adverse reactions such as cerebral edema and inflammation, and the therapeutic efficacy of the antibody is also under debate.

[0006] Therefore, there remains a strong need in the field to identify specific Aβ oligomers that play an important role in the development and progression of AD, so that they can be used to treat and / or prevent AD. Summary of the Invention

[0007] A first aspect of the present invention relates to an isolated Aβo*3F, wherein the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody or a variant thereof, and has a total molecular weight of approximately 588 kDa based on analysis by size exclusion chromatography (SEC), and the light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are set forth in SEQ ID NO:17-22, respectively, and the variant is a K98R variant or a 7B antibody, and the light chain CDR sequence and heavy chain CDR sequence are set forth in SEQ ID NO:25-30 or SEQ ID NO:32-35 and KAS, respectively.

[0008] In some embodiments, the isolated Aβo*3F is produced in vitro.

[0009] A second aspect of the present invention relates to a composition comprising Aβo*3F as described above.

[0010] In some embodiments, the composition is an immunogenic composition, a pharmaceutical composition, or a vaccine.

[0011] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient and / or adjuvant.

[0012] In some embodiments, the composition further comprises another active ingredient. Preferably, the other active ingredient is other forms of Aβ oligomers, protofibrils, and mature fibrils, such as Aβ dimers, trimers, hexamers, dodecamers, amylospheroids (ASPDs), amyloid beta-derived diffusible ligands (ADDLs), etc.; preferably, the other active ingredient is a binding agent that specifically binds to Aβo*3F. Preferably, the binding agent that specifically binds to Aβo*3F is 3F, K98R, or 7B. More preferably, the binding agent that specifically binds to Aβo*3F and the Aβo*3F are in separate states for simultaneous or sequential administration. In some embodiments, the composition further comprises another drug for treating mild cognitive impairment (MCI) or AD.

[0013] A third aspect of the present invention relates to a method for producing Aβo*3F in vitro, comprising the steps of: dissolving Aβ42, Aβ40, or other forms of Aβ in 50 mM NaOH to a concentration of 1 mg / mL, vortexing for 3-5 minutes, sonicating for 1 minute, and then diluting the solution to 10 μM with pre-chilled PBS; centrifuging the solution at 21,000 g at 4°C for 30-40 minutes and discarding the precipitate (approximately 5% of the initial volume) to obtain Aβ monomers; incubating the Aβ monomers at 25°C for 2 days under static conditions and then incubating them with 3F antibody-crosslinked Protein A magnetic beads at 4°C overnight; and on the second day, washing the magnetic beads three times with 0.1% PBST, eluting them with 20-100 mM glycine (pH 2.0) for 3-5 minutes, repeating the above elution twice, and neutralizing the eluate to pH 7 with 1 M Tris to obtain Aβo*3F.

[0014] A fourth aspect of the present invention relates to the use of Aβo*3F as a target in the manufacture of a medicament for preventing and / or treating MCI and / or AD in a subject, wherein the Aβo*3F is an Aβ oligomer that specifically binds to a 3F antibody or a variant thereof, and has a total molecular weight of approximately 588 kDa based on analysis by size exclusion chromatography (SEC), and the light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are set forth in SEQ ID NO:17-22, respectively, and the variant is a K98R variant or a 7B antibody, and the light chain CDR sequence and heavy chain CDR sequence are set forth in SEQ ID NO:25-30 or SEQ ID NO:32-35 and KAS, respectively.

[0015] In some embodiments, the drug is an antiserum, antibody, or small molecule drug that specifically binds to Aβo*3F, and preferably, the antibody is a polyclonal antibody or a monoclonal antibody or an antigen-binding fragment thereof.

[0016] Another aspect of the invention relates to a binding agent that specifically binds to Aβo*3F as described above, wherein said binding agent is 3F, K98R and 7B.

[0017] In some embodiments, the binding agent is an antiserum, antibody, or small molecule drug that specifically binds to Aβo*3F, and preferably the antibody is a polyclonal or monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a single-chain antibody. In some embodiments, the heavy chain CDR sequences of the antibody are set forth in SEQ ID NOs:25-27, respectively, and the light chain CDR sequences are set forth in SEQ ID NOs:28-30, respectively.

[0018] In another aspect, the present invention relates to a method for producing such a binding agent, the method comprising the step of immunizing an animal with an immunologically effective amount of such Aβo*3F, or screening a phage-displayed peptide library, antibody library, compound library, etc. using such Aβo*3F as a substrate.

[0019] In some embodiments, the animal is a mouse, rat, guinea pig, rabbit, sheep, goat, monkey, horse, cow, alpaca, or non-human primate.

[0020] Another aspect of the present invention relates to a composition comprising the above-described binding agent or a binding agent obtained by the above-described method, preferably the composition further comprising the above-described Aβo*3F, more preferably the binding agent that specifically binds to Aβo*3F and the Aβo*3F are in separate states for simultaneous or sequential administration. In some embodiments, the composition further comprises another therapeutic agent for neurodegenerative diseases, preferably the other therapeutic agent for neurodegenerative diseases is selected from acetylcholinesterase inhibitors such as donepezil, galantamine, and rivastigmine, and aspartate receptor antagonists such as memantine.

[0021] In another aspect, the present invention relates to the use of Aβo*3F, a composition or a binding agent as described above in the manufacture of a medicament for preventing and / or treating MCI and / or AD in a subject.

[0022] In another aspect, the present invention relates to an Aβo*3F, composition or binding agent as described above for use as a medicament for preventing and / or treating MCI and / or AD in a subject.

[0023] Another aspect of the invention relates to a method for preventing and / or treating MCI and / or AD in a subject, said method comprising the step of administering to said subject a prophylactically and / or therapeutically effective amount of Aβo*3F, composition or binding agent as described above.

[0024] Another aspect of the present invention relates to the use of Aβo*3F as a target for diagnosing MCI and / or AD in a subject, wherein the Aβo*3F is an Aβ oligomer that specifically binds to a 3F antibody or a variant thereof in the subject, and has a total molecular weight of approximately 588 kDa based on size exclusion chromatography (SEC) analysis. The light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are set forth in SEQ ID NOs:17-22, respectively. The variant is a K98R variant or a 7B antibody, and the light chain CDR sequence and heavy chain CDR sequence are set forth in SEQ ID NOs:25-30 or SEQ ID NOs:32-35 and KAS, respectively. In other words, the Aβo*3F in a subject as described above may be used as a biomarker for diagnosing whether or not the subject has MCI and / or AD. It will be clear to one skilled in the art how to diagnose whether a subject has and / or is at risk for MCI and / or AD based on Aβo*3F as a biomarker. In some embodiments, the diagnosis may be performed using an antibody that specifically binds to Aβo*3F, such as 3F, K98R, or 7B.

[0025] Another aspect of the present invention relates to the use of Aβo*3F as a target for preventing and / or treating MCI and / or AD in a subject, wherein the Aβo*3F is an Aβ oligomer that specifically binds to a 3F antibody or a variant thereof in the subject, and has a total molecular weight of approximately 588 kDa based on size exclusion chromatography (SEC) analysis. The light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are set forth in SEQ ID NOs: 17-22, respectively. The variant is a K98R variant or a 7B antibody, and the light chain CDR sequence and heavy chain CDR sequence are set forth in SEQ ID NOs: 25-30 or SEQ ID NOs: 32-35 and KAS, respectively. In other words, Aβo*3F in a subject as described above may be a target for preventing and / or treating MCI and / or AD in the subject. It will be clear to those skilled in the art how to prevent and / or treat MCI and / or AD in a subject based on Aβo*3F as a target. In some embodiments, the prevention and / or treatment may be carried out using an antibody that specifically binds to Aβo*3F, such as 3F, K98R or 7B, or a small molecule drug.

[0026] In some embodiments, the subject is a human, a non-human primate, a cat, or a dog.

[0027] In other words, in previous research, the inventors were the first in the world to screen for a fully human single-chain antibody, W20 (CN101463082A), which specifically binds to Aβ oligomers using phage display technology. The 3F antibody (amino acid sequence shown in SEQ ID No. 23) is an improved version of the W20 antibody, and has significantly improved affinity for Aβ oligomers compared to the W20 antibody. It can more significantly inhibit Aβ aggregation and Aβ oligomer-induced neuronal toxicity, 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 in the Aβ oligomer mixture, with potent pathogenic effects and an important role in the development and progression of AD. The highly toxic oligomers recognized by 3F are present in the cerebrospinal fluid (CSF), blood, and / or brain tissue of AD patients and AD-derived MCI patients, and their levels show marked differences in the CSF, blood, and / or brain tissue of three types of humans: AD patients, MCI patients, and healthy elderly individuals, allowing for accurate differentiation between AD patients, MCI patients, and healthy elderly individuals. These toxic oligomers are also present in AD transgenic mice and are directly related to the onset of AD transgenic mice. In the present invention, the highly toxic Aβ oligomer recognized by 3F is called AβO*3F. It can be isolated from Aβ oligomer mixtures by immunoprecipitation (using 3F antibodies). Its typical characteristics are 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). Its toxicity to neurons is more than 200-fold greater than that of Aβ oligomer mixtures. AβO*3F can activate microglia and / or astrocytes to secrete large amounts of proinflammatory cytokines. More importantly, the present invention produces AβO*3F in vitro, and the composition of the AβO*3F produced in vitro is identical to that of the product immunoprecipitated from cerebrospinal fluid or plasma of AD patients using 3F antibodies, and it has the same physicochemical properties and functions.The Aβo*3F produced in vitro may be used, for example, as an immunogen to immunize a target organism to obtain antisera, polyclonal antibodies, or monoclonal antibodies against Aβo*3F, which may be used to treat and / or prevent AD or MCI patients, or may be used, for example, as a therapeutic or prophylactic vaccine to immunize a target patient and treat and / or prevent AD or MCI in the target patient. The Aβo*3F or compositions thereof described herein provide a new therapeutic and / or prophylactic target and a new therapeutic and / or prophylactic route for AD or MCI. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram showing the production of Aβo*3F, Aβ*6E10, and Aβ-ID by immunoprecipitation.

[0029] [Figure 2]Figure 2 shows the molecular weight and morphological characteristics of Aβo*3F. Figure 2A: Detection of Aβ aggregation state by ThT; Figure 2B: Detection of the binding status of oligomers obtained under different incubation conditions with 3F, A11, and 6E10 antibodies by dot blot; Figure 2C: Affinity of Aβos obtained by incubation with 10 μM Aβ for 0 to 4 days with 3F antibody; Figure 2D: Detection of the band distribution of sAβo*3F by Western blot; Figure 2E: Analysis of the molecular weight of sAβo*3F by size exclusion chromatography (SEC); SEC analysis of sAβo*3F and SEC standard (Superdex 200) 10 / 300), 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); Figure 2F: Linear equation (Y) and correlation coefficient (r2) fitted based on the molecular weight and elution volume of SEC standards; Figure 2G: Detection of band distribution of mAβo*3F by Western blot; Figure 2H: Analysis of molecular weight of mAβo*3F by SEC; Figure 2I: Detection of band distribution of hAβo*3F by Western blot.

[0030] [Figure 3] Figure 3 shows the toxic effects of Aβo*3F on neuronal cells. Figure 3A: IC50 of sAβos toxicity on N2a cells; Figure 3B: IC50 of sAβo*3F toxicity on N2a cells; Figure 3C: Comparison of the neurotoxicity of sAβos, sAβo*3F, and sAβ-ID on N2a cells by MTT assay; Figure 3D: IC50 of mAβo*3F toxicity on N2a cells and primary neurons; Figure 3E: Comparison of the cytotoxicity of mAβ*6E10, mAβo*3F, and mAβ-ID on primary neurons by MTT assay; Figure 3F: IC50 of hAβo*3F toxicity on primary neurons; Figure 3G: Comparison of the cytotoxicity of hAβ*6E10, hAβo*3F, and hAβ-ID on primary neurons by MTT assay.

[0031] [Figure 4] These figures show the effects of mAβo*3F on the expression levels of cytokines in glial cells. Figure 4A shows the effects of mAβo*3F on the expression levels of TNF-α, IL-1β, and IL-6 in microglia; Figure 4B shows the effects of mAβo*3F on the expression levels of TNF-α, iNos, IL-1β, and IL-6 in primary astrocytes; Figure 4C shows the effects of mAβo*3F on the expression levels of TSP1, Gpc4, and Gpc6 in primary astrocytes.

[0032] [Figure 5] Figure 5 shows the effects of Aβo*3F on cognition and neuronal damage in mice. Figure 5A shows the cognitive scores of each group of mice in the novel object recognition experiment; Figure 5B shows the duration of time in the novel arm of each group of mice in the Y-maze experiment; Figure 5C shows the dendritic spine density of mouse neurons detected by Golgi staining; Figure 5D shows statistical analysis of the dendritic spine density in Figure 5C; Figure 5E shows the number of hippocampal neurons detected by Ziehl-Neelsen staining (scale bar: 20 μm); Figure 5F and G show statistical analysis of the number of neurons in the DG (F) and CA1 (G) regions using Image J software.

[0033] [Figure 6] Figure 6 shows that Aβo*3F activates glial cells in the mouse brain and induces neuroinflammation. Figure 6A: Immunohistochemical detection of microglial activation in the DG and CA1 regions of the mouse hippocampus (scale bar: 20 μm). Figure 6B: Immunohistochemical detection of astrocyte activation in the DG and CA1 regions of the mouse hippocampus (scale bar: 20 μm). Figures 6C and 6D: Quantification of the area of Iba-1-positive microglia in the DG (C) and CA1 (D) regions using Image J software. Figures 6E and 6F: Quantification of the area of GFAP-positive astrocytes in the DG (E) and CA1 (F) regions using Image J software. Figures 6G and 6H: ELISA detection of IL-6 (G) and IL-1β (H) expression levels in the mouse hippocampus.

[0034] [Figure 7] 7 shows the detection of Aβo*3F levels in CSF and plasma of AD patients by electrochemiluminescence. Figures 7A and 7B: Detection of corresponding Aβ42 (A) and Aβ40 (B) levels in Aβo*3F isolated from CSF of AD patients and a healthy control group (Con) by MSD; Figures 7C and 7D: Detection of corresponding Aβ42 (C) and Aβ40 (D) levels in Aβo*3F isolated from plasma of mild cognitive impairment (MCI), AD, and a healthy control group (Con) by MSD.

[0035] [Figure 8]

[0043] Figure 1 shows Aβ oligomers that specifically bind to antibodies induced by the ABW vaccine. Blood was collected from the animals two weeks after the third and fourth immunizations, and the antibody titers in the serum were measured by ELISA.

[0036] [Figure 9] This figure shows that ABW vaccine significantly improved memory in AD transgenic mice in a water maze experiment: (A) During the training period, the latency of the mice to find the platform; (B-D) After the platform was removed, the latency of the mice to reach the platform area (B), the number of times they crossed the platform location (C), and the time spent in the target quadrant (D).

[0037] [Figure 10] Figure 1 shows the reduction of Aβ levels in the brains of AD mice treated with the ABW vaccine. The levels of soluble Aβ40 (A), soluble Aβ42 (B), and insoluble Aβ40 and Aβ42 (C) in the brains of AD mice treated with the ABW vaccine were measured by ELISA.

[0038] [Figure 11]1 shows a reduction in Aβ plaques in the brains of AD mice by ABW vaccination, immunohistochemical staining was performed with 6E10 antibody, and the scale bar is 100 μm; the stained area of Aβ plaques was quantitatively analyzed.

[0039] [Figure 12] FIG. 1 shows the results of ELISA for the binding of antibody 7B to Aβo*3F.

[0040] [Figure 13] FIG. 1 shows the cognitive index of mice in each group in a novel object recognition experiment, demonstrating that the 7B antibody significantly improves the cognitive ability of AD transgenic mice.

[0041] [Figure 14] FIG. 1 shows the reduction of Aβ plaques in the brains of AD mice by 7B antibody as determined by thioflavin S (THS) staining.

[0042] [Figure 15] This figure shows that the binding affinity to Aβo*3F is further improved after K at position 98 of the heavy chain of the 3F antibody is mutated to R, suggesting that the combination of this single-chain antibody with Aβ oligomers may have better activity. DETAILED DESCRIPTION OF THE INVENTION

[0043] Definition: Unless otherwise stated, terms used in the claims and specification are defined as follows: Unless otherwise defined herein, scientific and technical terms used in connection with the methods and compositions of the invention described herein shall have the ordinary meanings understood by those of ordinary skill in the art. Also, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature used in connection with and the following techniques are those well known and commonly used in the art: biochemistry, immunology, enzymology, molecular and cell biology, microbiology, genetics, and polypeptide chemistry described herein.

[0044] Unless otherwise specified, the methods and techniques described herein are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed herein. 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 until 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 University 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).

[0045] All publications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise indicated, the following terms shall be understood to have the following meanings: When used in conjunction with a numerical value, the term "about" refers to any numerical value within ±1, ±5, or ±10% of the referenced numerical value.

[0046] The term "Aβo*3F" refers to an Aβ oligomer that specifically binds to the 3F antibody, and has a molecular weight of approximately 588 kDa based on size exclusion chromatography (SEC) analysis, and the light chain CDR sequences and heavy chain CDR sequences of the 3F antibody are set forth in SEQ ID NOs: 17-22, respectively. In some embodiments, the size exclusion chromatography (SEC) analysis is performed using a Superdex200 10 / 300 GL molecular sieve column.

[0047] In some embodiments, the Aβo*3F is isolated Aβo*3F. In some embodiments, the Aβo*3F is produced in vitro. In some embodiments, the Aβo*3F is produced in vitro and purified. In some embodiments, the purity of the purified Aβo*3F is at least 1%, for example, 1.5 to 99.9%, preferably 15% to 99.9%, 25% to 99.9%, 50% to 99.9%, 65% to 99.9%, 95% to 99.9%, or 95% to 99.9%, for example, at least 2%, 3%, 4%, The Aβo*3F purification efficiency is 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher. In some embodiments, the purification is affinity purification, e.g., using a monoclonal antibody specific for Aβo*3F, e.g., 3F. In some embodiments, Aβo*3F is produced in vitro as follows.

[0048] Aβ42, Aβ40, or other forms of Aβ were dissolved in 50 mM NaOH at a concentration of 1 mg / mL, vortexed for 3–5 min, sonicated for 1 min, and then diluted to 10 μM with pre-chilled PBS. The solution was centrifuged at 21,000 g for 30–40 min at 4°C, and the precipitate (approximately 5% of the initial volume) was discarded to obtain Aβ monomers. The Aβ monomers were incubated at 25°C for 2 days under static conditions and then incubated overnight at 4°C with protein A magnetic beads cross-linked with 3F antibody. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20–100 mM glycine (pH 2.0) for 3–5 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβo*3F.

[0049] In some embodiments, provided herein are compositions comprising Aβo*3F, the weight of which is between 0.01% and 99.9% of the total weight of the composition, e.g., 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the compositions provided herein contain Aβo*3F, wherein the amount of Aβo*3F present in the composition is typically 1 to 200 μg / mL, preferably 1 to 100 μg / mL, and preferably 5 to 50 μg / mL, preferably 8 to 40 μg / mL, and more preferably 16 to 32 μg / mL, for example, 16 μg / mL, 18 μg / mL, 20 μg / mL, 22 μg / mL, 24 μg / mL, 26 μg / mL, 28 μg / mL, 30 μg / mL, or 32 μg / mL. A suitable dosage for humans is typically 0.25 to 1.5 mL in volume. In one embodiment, the dosage for humans is 0.5 mL. In a further embodiment, the dosage for humans is greater than 0.5 mL, for example, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1 mL. In a further embodiment, the dosage for humans is 1 mL to 1.5 mL.

[0050] In some embodiments, the composition is an immunogenic composition. As used herein, an "immunogenic composition" refers to a composition capable of generating an immune response in a host or subject to which the composition is administered. An immunogenic composition comprises an immunologically effective amount of the AβO*3F of the present invention and any other components. An "immunologically effective amount" means that administration of that amount to an individual, either as a single dose or as part of a series of doses, is therapeutically or prophylactically effective or capable of producing a detectable amount of polyclonal antibodies or polyclonal antibodies. This amount will vary depending on the health and physical condition of the individual being treated, their age, the degree of protection desired, the vaccine formulation, and other relevant factors. This amount falls within a relatively broad range that can be determined by routine testing.

[0051] In some embodiments, the immunogenic composition may further comprise a pharmaceutically acceptable carrier. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The immunogenic compositions of the present invention may be formulated as pharmaceutical compositions before administration to a subject. The present invention further provides a vaccine comprising the immunogenic composition of the present invention and a pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered with the composition and is non-toxic and does not interfere with the effectiveness of the active ingredient. Vaccine formulations are generally described in Vaccine Design ("The subunit and adjuvant approach" (eds. Powell MF & Newman MJ) (1995) Plenum Press New York).

[0052] The immunogenic composition or vaccine of the invention may be included in a container, package or dispenser together with instructions for administration.The invention provides a kit comprising a first container (i) containing the immunogenic composition or vaccine of the invention, and optionally a second container (ii) containing an adjuvant as described herein. The present invention further provides compositions, which optionally further comprise other forms of Aβ oligomers, protofibrils, and mature fibrils, such as Aβ dimers, trimers, hexamers, dodecamers, ASPDs, ADDLs, etc.

[0053] In some embodiments, a composition described herein (e.g., a pharmaceutical composition and / or an immunogenic composition) comprises or is used in combination with an adjuvant. An adjuvant for use with a composition described herein may be administered before (e.g., within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 10 minutes), simultaneously with, or after (e.g., within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, or 10 minutes) administration of the composition. In some embodiments, the compositions described herein do not include or are not used in combination with an adjuvant.

[0054] The immunogenic compositions disclosed herein typically include one or more pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers and excipients are well known and may be selected by those skilled in the art. The adjective "pharmaceutically acceptable" indicates that the subject matter is suitable for administration to a subject (e.g., a human or animal subject). Remington's P Harmaceutical Sciences, E.W. Martin, Mack Publishing Co., Easton, Pa., 15th ed. (1975) describes compositions and formulations (including diluents) suitable for drug delivery therapeutic and / or prophylactic compositions, including immunogenic compositions.

[0055] For example, the carrier or excipient may suitably comprise a buffer. Optionally, the carrier or excipient further comprises at least one component that stabilizes solubility and / or stabilizes the carrier or excipient. Examples of solubilizers / stabilizers include soil release agents, such as lauroyl sarcosine and / or Tween. Many pharmaceutically acceptable carriers and / or pharmaceutically acceptable excipients are known in the art and can be found in, for example, Remington's P and others, such as those described in "Pharmaceutical Sciences," E. W. Martin, Mack Publishing Co., Easton, Pa., 5th Edition (1975). Thus, one skilled in the art can select appropriate excipients and carriers to prepare a formulation suitable for delivery to a subject via a selected route of administration.

[0056] Suitable excipients include, but are not limited to, glycerin, polyethylene glycol (PEG), sorbitol, trehalose, N-lauroyl sarcosine sodium salt, L-proline, the non-soil release agent sulfobetaine, guanidine hydrochloride, urea, trimethylamine oxide, KCl, salts of Ca2+, Mg2+, Mn2+, Zn2+ and other divalent cations, dithiothreitol, dithioerythritol, and 13-mercaptoethanol. Other excipients may be stain release agents (including Tween 80, Tween 20, Triton X-00, NP-40, Empigen BB, octyl glucoside, lauroyl maltoside, Zwittergent 3-08, Zwittergent 3-0, Zwittergent 3-2, Zwittergent 3-4, Zwittergent 3-6, CHAPS, sodium deoxycholate, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide). Optionally, the immunogenic compositions of the invention can be prepared to include other components, such as adjuvants, stabilizers, pH adjusters, preservatives, and the like.

[0057] As used herein, "adjuvant" refers to a composition that enhances the immune response to an immunogen. Compositions containing the adjuvants of the present invention may be used, for example, as vaccines in human subjects. Adjuvants accelerate, prolong, and / or enhance the quality and / or strength of the immune response to an antigen / immunogen compared to administration of the antigen alone, thereby reducing the amount of antigen / immunogen required for any given vaccine and / or the frequency of injections required to generate an adequate immune response to the antigen / immunogen of interest.

[0058] In the context, examples of adjuvants that can be used in the compositions of the invention include inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide); gel-like precipitates of aluminum hydroxide (alum); AlPO4; hydroxygel; bacterial products derived from the outer membrane of Gram-negative bacteria, in particular monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), muramyl dipeptide and derivatives thereof; Freund's incomplete adjuvant; liposomes, in particular neutral liposomes, liposomes containing this composition and any cytokine; AS01B, AS01E, AS02; nonionic block copolymers; ISCOMATRIX adjuvants, unmethylated DNA containing CpG dinucleotides (CpG motifs), in particular CpG ODN having a phosphorothioate (PTO) backbone (CpG PTO ODN) or a phosphodiester (PO) backbone (CpG PO ODN). Adjuvants include ODNs; synthetic lipopeptide derivatives, particularly Pam3Cys; lipoarabinomannan; peptidoglycan; zymosan; heat shock proteins (HSPs), particularly HSP 70; dsRNA and its synthetic derivatives, particularly poly I:poly C; polycationic peptides, particularly poly-L-arginine; paclitaxel; fibronectin; flagellin; imidazoquinolines; cytokines with adjuvant activity, particularly GM-CSF, interleukins (IL-2, IL-6, IL-7, IL-18), type I and type II interferons, particularly interferon-γ and TNF-α; 2,5-dihydroxyvitamin D3 (calcitriol); and synthetic oligopeptides, particularly peptides presented by MHC II. Nonionic block copolymers containing polyoxyethylene (POE) and polyoxypropylene (POP), such as POE-POP-POE block copolymers, may also be used as adjuvants.

[0059] Further examples of adjuvants include inorganic adjuvants (including, for example, inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (including, for example, saponins such as QS21 or squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immune stimulating complexes (ISCOMS), liposomes, etc.), and the like. These include adjuvants such as oocytes, biodegradable microspheres, virions, bacterial adjuvants (e.g., monophosphoryl lipid A such as 3-deacylated-O-monophosphoryl lipid A (3D-MPL) and muramyl peptides), synthetic adjuvants (e.g., monophosphoryl lipid A (MPL), particularly 3-deacylated-O-monophosphoryl lipid A (3D-MPL)), muramyl peptide analogs or synthetic lipid A, and synthetic polynucleotide adjuvants such as polyarginine or polylysine.

[0060] Saponins are also suitable adjuvants, such as the saponin Quil-A (derived from the bark of the South American Quillaja Molina tree) and its fractions. Purified fractions of Quil-A, also known as immunostimulants, include squalene, QS21, QS17, and QS7 (the non-hemolytic fraction of Quil-A). Combinations of QS21 with polysorbates or cyclodextrins are also suitable.

[0061] Another example of an adjuvant is an immunostimulatory oligonucleotide that contains unmethylated cytosine-guanine dinucleotide sequences ("CpG") present in DNA. When administered by systemic and mucosal routes, CpG is called an adjuvant. When formulated into a vaccine, it may be administered in free solution with free antigen, or covalently bound to the antigen, or formulated with a carrier such as aluminum hydroxide. Activation of certain receptors may stimulate immune responses. Such receptors are known to those skilled in the art and include, for example, cytokine receptors, particularly type I cytokine receptors, type II cytokine receptors, TNF receptors; vitamin D receptors that function as transcription factors; Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9. Agonists of such receptors have adjuvant activity, i.e., immunostimulatory properties. Other suitable adjuvants include aminoalkyl glucosaminide phosphates (AGPs) or pharmaceutically acceptable salts of AGPs. Some AGPs are TLR4 agonists and some are TLR4 antagonists. The adjuvant of the composition of the present invention may be one or more Toll-like receptor agonists. In a more preferred embodiment, the adjuvant is a Toll-like receptor 4 agonist. In a particularly preferred embodiment, the adjuvant is a Toll-like receptor 9 agonist.

[0062] Such adjuvants may be formulated with carriers such as liposomes, oil-in-water emulsions and / or metallic salts (including aluminum salts such as aluminum hydroxide). For example, 3D-MPL may be formulated with aluminum hydroxide or an oil-in-water emulsion, QS21 may be formulated with cholesterol-containing liposomes, oil-in-water emulsions or alum, and CpG may be formulated with alum or other cationic carriers.

[0063] Adjuvant combinations that can be used in the present invention include, in particular, combinations of monophosphoryl lipid A and saponin derivatives, more particularly, combinations of QS21 and 3D-MPL, or compositions in which QS21 is quenched in cholesterol-containing liposomes (DQ). Alternatively, combinations of CpG and saponins (e.g., QS21) are suitable adjuvants for use in the present invention, as are effective adjuvant formulations of QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion. Saponin adjuvants may also be formulated in liposomes and combined with immunostimulatory oligonucleotides. Thus, suitable adjuvant systems include, for example, combinations of monophosphoryl lipid A (preferably 3D-MPL) and aluminum salts. Other exemplary adjuvants include QS21 and / or MPL and / or CpG. QS21 may also be quenched in cholesterol-containing liposomes.

[0064] The present invention further provides a method for producing the immunogenic composition or vaccine of the present invention, said method comprising the step of mixing the AβO*3F of the present invention with a pharmaceutically acceptable excipient or carrier. The compositions of the present invention may be in aqueous form (i.e., solution or suspension) or in dry form (e.g., lyophilized form). When a dried vaccine is used, the liquid medium is reconstituted before injection. Freeze-drying of vaccines is known in the art. When the immunogenic composition of the present invention comprises lyophilized components, the components are typically prepared separately, mixed, and then lyophilized. To stabilize the antigen during lyophilization, inactive ingredients may be added before lyophilization, e.g., as stabilizers. Preferred stabilizers for inclusion are lactose, sucrose, mannitol, and mixtures thereof, such as lactose / sucrose mixtures, sucrose / mannitol mixtures, etc. Thus, the final vaccine obtained by aqueous reconstitution of the lyophilized material may contain lactose and / or sucrose. Preferably, amorphous excipients and / or amorphous buffers are used when preparing lyophilized vaccines.

[0065] In some embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein (e.g., drugs and / or immunogenic compositions) may be formulated for subcutaneous, parenteral, oral, sublingual, buccal, intradermal, transdermal, colorectal, intraperitoneal, rectal, intravenous, intranasal, intratracheal, intramuscular, topical, transdermal, or intradermal administration. In a specific embodiment, the compositions provided herein (e.g., drugs and / or immunogenic compositions) are formulated for intramuscular injection. In some embodiments, the Aβo*3F described herein may be used to prevent and / or treat AD, for example, as a vaccine to induce an immune response. As used herein, induction of an immune response refers to the ability of the Aβo*3F (also referred to as an "antigen" or "immunogen") to induce a T cell and / or humoral immune response to Aβo*3F. For example, an immunogenic composition can induce memory T and / or B cell populations relative to untreated subjects following immunization with the composition. Immune responses may be measured by methods known in the art, including measuring proliferation or induction of effector function of specific lymphocyte types of interest (e.g., B cells, T cells, T cell lines and T cell clones).

[0066] Thus, in some embodiments, there is provided a method of inducing an immune response in a subject, the method comprising administering to the subject Aβo*3F or an immunogenic composition of the present invention. The immune response is preferably protective, and preferably involves antibodies. The method may also result in an enhanced response. The compositions of the present invention are preferably administered to a patient in a 0.5 mL dose (as described above). In one embodiment, the subject is Aβo*3F seronegative. A subject is "seronegative" if the subject has no past or present serological evidence of the presence of Aβo*3F. In another embodiment, the subject is Aβo*3F seropositive. A subject is "seropositive" if the subject has past or present serological evidence of the presence of Aβo*3F.

[0067] The present invention further provides dosing regimens that maximize the immunogenicity of the Aβo*3F or immunogenic compositions of the invention by design. Thus, in one embodiment, a method of inducing an immune response in a subject is provided, comprising administering two or more doses of the Aβo*3F and / or immunogenic compositions of the invention to the subject. In some embodiments, the doses are spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more apart. In another embodiment, the doses are spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more apart. Alternatively, the doses may be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years or more apart.

[0068] The present invention further provides a method for treating and / or preventing AD in a subject, said method comprising the step of administering to said subject Aβo*3F or an immunogenic composition of the present invention. In some embodiments, a composition or AβO*3F described herein may be administered to a subject to induce an immune response, including antibody production. Such antibodies may be isolated by techniques known to those skilled in the art (e.g., immunoaffinity chromatography, immunoprecipitation, centrifugation, etc.).

[0069] The ability of AβO*3F or compositions described herein to generate an immune response in a subject may be assessed by any method known to one of skill in the art or described herein, for example, by an immunoassay such as an ELISA (see, e.g., Van den Dobbelsteen et al., 2016, Vaccine 34:4152-4160) or an electrochemiluminescence- or chemiluminescence-based immunoassay.

[0070] Antibodies induced, elicited, or identified by the AβO*3F or compositions provided herein may be used to monitor the effectiveness of therapy and / or disease progression. Any immunoassay system known to those skilled in the art may be used for this purpose, including, but not limited to, competitive and non-competitive assay systems using the following techniques: radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence- or chemiluminescence-based immunoassay, "sandwich" immunoassay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, immunoradiometric assay, fluorescence immunoassay, protein A immunoassay, and immunoelectrophoresis assay. Some of these assays, such as electrochemiluminescence- or chemiluminescence-based immunoassay, can be performed in a multiplex format, and a multiplex assay format is usually preferred.

[0071] The present invention further provides AβO*3F or a composition of the present invention for use as a medicament. The present invention further provides the use of AβO*3F or a composition of the present invention in the manufacture of a medicament for raising an immune response in a mammal. These uses and methods are preferably used to prevent and / or treat MCI and / or AD in a subject.

[0072] In some embodiments, AβO*3F or compositions of the invention may be used to diagnose MCI and / or AD in a subject. As used herein, the term "treatment" refers to therapeutic treatment and prophylactic measures, where the goal is to prevent or slow (alleviate) undesirable physiological changes or disorders, such as the progression of AD. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation and remission of disease state (whether partial or complete, detectable or undetectable). "Treatment" can also mean extending survival beyond that expected in the absence of treatment. Those in need of treatment include those suffering from a disease or disorder, those susceptible to a disease or disorder, and those in whom the onset of a disease or disorder is to be prevented. As used herein, a "drug" is an agent for treating an undesirable physiological change or disorder.

[0073] The term "antiserum" refers to serum containing polyclonal antibodies. In some embodiments, the antiserum is obtained by immunizing a target organism, such as a mammal, including a mouse, rat, guinea pig, rabbit, monkey, goat, sheep, horse, or cow, with AβO*3F or a composition described herein.

[0074] The term "polyclonal antibody" refers to a mixture of multiple antibodies produced when multiple antigenic determinants of an antigen stimulate the body, i.e., a polyclonal antibody. The AβO*3F described herein is a complex protein oligomer, and antibodies produced by immunizing animals with this oligomer are typically polyclonal. Further antibodies against individual antigenic determinants can be isolated and purified from the polyclonal antibodies produced, for example, by affinity chromatography, immunoprecipitation, size exclusion chromatography, etc. The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule capable of binding to an epitope of an antigen. Naturally occurring antibodies typically comprise a tetramer and usually consist of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the IgG, IgA, IgM, IgD, and IgE isotypes, with the corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Ig antibodies of the same class may be divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and location of disulfide bonds in the heavy chain. For example, IgG may be divided into subtypes of IgG1, IgG2, IgG3, and IgG4, and IgA may be divided into subtypes of IgA1 and IgA2. Light chains are divided into κ and λ chains depending on the constant region. Antibodies of the present invention may have any isotype. The choice of isotype is usually determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the κ or λ human light chain constant region can be used. If necessary, the class of the antibody of the present invention can be converted using known methods. For example, the initial IgG antibody of the present invention can be class-converted to the IgM antibody of the present invention. Furthermore, class conversion techniques can be used to convert an IgG subclass to another subclass, for example, IgG1 to IgG2.Therefore, if the effector function of the antibodies of the present invention can be converted to, for example, an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody by isotype switching, and the C1q binding activity of the antibodies of the present invention is reduced or eliminated, the antibodies can be used for various therapeutic purposes. In some embodiments, the antibodies of the present invention are IgM, IgG1, IgG2, IgG3, or IgG4 antibodies. An antibody belongs to a particular isotype if its amino acid sequence is nearly identical to that of another isotype.

[0075] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen-binding site and includes natural and artificial antibodies of various structures, including, but not limited to, complete antibodies and antigen-binding fragments of antibodies.

[0076] A "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved 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), and the heavy chain constant region is usually 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 regions 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 binding regions that interact with antigens. The VH and VL regions may be further subdivided into hypervariable regions (HVRs) called "complementarity-determining regions (CDRs)," with more conserved regions called "framework regions" (FRs) intervening between them. Each VH and VL is composed of three CDR regions and four FR regions, arranged from the amino terminus to the carboxyl terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0077] The terms "complementarity determining region" or "CDR region" or "CDR" (which may be used interchangeably herein with hypervariable region "HVR") refer to the regions of an antibody variable domain that form highly variable sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to an epitope of an antigen. Herein, the three CDRs of a heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of a light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0078] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different numbering schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined by different numbering schemes will be different. Therefore, when an antibody is defined using a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but whose so-called CDR boundaries differ from the specific CDR boundaries defined in the present invention by applying a different scheme (e.g., a different numbering scheme rule or combination).

[0079] The terms "monoantibody," "monoclonal antibody," or "monoclonal antibody composition" refer to a preparation of antibody molecules of single molecular composition, derived from a population of essentially homogeneous antibodies; i.e., a population comprising individual antibodies that are identical except for any naturally occurring mutations that may be present. A typical monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. In some embodiments, a monoclonal antibody may be composed of two or more Fab domains, thereby increasing its specificity for two or more targets. The terms "monoclonal antibody" or "monoclonal antibody composition" are not limited to any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).

[0080] The present invention further includes "bispecific antibodies." The term "antigen-binding fragment of an antibody" refers to a fragment, portion, region, or domain of an antibody (which may be obtained, for example, by truncation, recombinantly, synthetically, etc.) that is capable of binding to an epitope. An antigen-binding fragment may comprise one, two, three, four, five, or all six CDR regions of such an antibody, and may exhibit different specificities, affinities, or selectivities while still being capable of binding to said epitope. Preferably, the antigen-binding fragment comprises all six CDR regions of said antibody. An antigen-binding fragment of an antibody may be part of or comprise a single polypeptide chain (e.g., an scFv), or may comprise two or more polypeptide chains (each having an amino terminus and a carboxyl terminus), such as a bibody, an Fab fragment, an Fab' fragment, or a Fab' fragment. F(ab')2 fragment, Fd fragment, Fv fragment, dAb fragment Examples include antibodies against antibodies against guanine, camelids, or nanobodies.

[0081] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are single chain antibodies. In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are chimeric antibodies. In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are humanized antibodies. In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are human or fully human antibodies. Variant antibodies are also included within the scope of the present invention.

[0082] The amino acid residues herein are numbered according to IMGT (registered trademark) (the international ImMunoGeneTics information system) (registered trademark) 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, United States 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 amino acid residues herein are numbered according to the Kabat numbering system.

[0083] An antibody or antigen-binding fragment thereof "specifically" binds to a region (i.e., epitope) of another molecule if it reacts or binds to the epitope more frequently, more rapidly, with a longer duration, and / or with greater affinity than other epitopes. In some embodiments, an antibody or antigen-binding fragment thereof of the invention binds to an amyloid protein, particularly its toxic form, particularly AβO*3F, with an affinity of at least 10 M, e.g., 10 M, 10 M, 10 M, 10 M, or higher. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, specific binding to an amyloid protein, particularly its toxic form, particularly AβO*3F, refers to the ability of the antibody or antigen-binding fragment thereof to bind to an amyloid protein, particularly its toxic form, particularly AβO*3F, with the above-mentioned specificity and / or under such conditions. Suitable methods for determining such binding are known in the art.

[0084] In the context of antibody binding to a designated antigen, the term "binding" typically refers to binding with an affinity corresponding to a KD of about 10-6 M or less, which KD is at least 10-fold, e.g., at least 100-fold, or at least 1,000-fold lower than the affinity of the antibody binding to a nonspecific antigen other than the designated antigen or a closely related antigen (e.g., BSA, casein).

[0085] As used herein, the term "kd" (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value. As used herein, the term "ka" (M-1 x sec-1 or 1 / Msec) refers to the association rate constant of a particular antibody-antigen interaction. As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing kd by ka. As used herein, the term "K" (M-1 or 1 / M) refers to the binding equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing k by k.

[0086] Antibodies of the present invention can be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, used alone or in combination. Typically, the amino acid sequence of the desired sequence is known, and one of skill in the art can readily produce the antibody using standard techniques for producing polypeptides. For example, these antibodies can be synthesized by known solid-phase methods, preferably using commercially available peptide synthesizers (e.g., those manufactured by Applied Biosystems, Foster City, California) according to the manufacturer's instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques known in the art. For example, a DNA sequence encoding the antibody can be incorporated into an expression vector and the vector introduced into a suitable eukaryotic or prokaryotic host for expression of the desired antibody, resulting in the antibody as a DNA expression product, which can then be isolated from the host using known techniques.

[0087] The antibodies and antigen-binding fragments thereof of the present invention can be modified by including any "suitable" number of modified amino acids and / or by attaching coupling substituents. The antibodies and antigen-binding fragments thereof of the present invention can also be chemically modified by covalent conjugation to a polymer to increase their circulating half-life.

[0088] 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, such as CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (e.g., 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). Antibodies of the invention and culture medium are suitably isolated by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0089] The term "subject" refers to a warm-blooded animal, preferably a mammal (human, domestic and farm animals, zoo animals, sporting or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc.), more preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting admission, receiving medical care, or the subject of a medical program or disease progression monitoring. In one embodiment, the subject is an adult (e.g., a subject 18 years of age or older). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is elderly, over 55, 60, 65, 70, 75, 80, 85, 90 years of age, or older. In one embodiment, the subject is male. In another embodiment, the subject is female.

[0090] The following examples are provided to illustrate the technical solutions of the present invention, but these examples are illustrative and illustrative and are not limiting. Unless otherwise noted, all reagents used in the following examples can be easily purchased from reagent companies such as Sigma-Aldrich and Merck. Unless otherwise noted, the test methods 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.

[0091] Example Example 1: Obtaining Aβ oligomers that specifically bind to 3F 1.1 Experimental materials and methods 1.1.1 Experimental materials Protein A magnetic beads: Bio-RAD, #1614833. Protein G magnetic beads: Bio-RAD, #1614023. Protein A / G agarose gel: Abmart, #A10001S. 6E10 antibody: Biolegend, #803002. Human-derived Aβ42 detection kit: Immuno-Biological Laboratories.

[0092] 1.1.2 Main solutions (1) 50 mM sodium hydroxide (pH 10.0). (2) 20 mM phosphate buffer solution (PBS): pH 7.4. (3) 0.1% PBST: PBS containing 0.1% Tween-20. (4) 20 mM glycine eluate (pH 2.0). (5) 1M Tris neutralization solution: pH 10.0. Note: Relevant reagents and solutions mentioned in Examples 2, 3, 4, 5 and 6, but whose origin is not indicated, are the same as in Example 1.

[0093] 1.2 Preparation of mouse brain homogenate APP / PS1 mice were deeply anesthetized with pentobarbital sodium and injected intracardially with ice-cold PBS containing 10 U / mL heparin. The mice were then sacrificed and their brains were removed. The brain tissue was then homogenized in 1 mL of RIPA strong lysis agent (containing protease and phosphatase inhibitors) using a Tissue Lyser II tissue homogenizer at 30 Hz for 8 min, then centrifuged at 14,000 rpm for 30 min at 4°C. The supernatant was collected. Prior to immunoprecipitation, the brain homogenate was loaded onto 100 μL of protein A / G-agarose gel and incubated at 4°C for 1 h to remove endogenous IgG. The brain homogenate was then loaded onto protein A magnetic beads cross-linked with an APP antibody to remove endogenous APP.

[0094] 1.3 Cross-linking of antibodies with magnetic beads First, the 3F and 6E10 antibodies were cross-linked to protein A or protein G magnetic beads, respectively. The specific cross-linking steps are as follows: (1) 200 μL of Protein A or Protein G magnetic beads were taken and washed three times with 2 mL of 0.1% PBST, with the magnetic beads being thoroughly mixed each time to homogenize them, and then placed on a magnetic shelf. The supernatant was discarded. (2) 50 μg of 3F or 6E10 antibody was added to the magnetic beads, and the mixture was shaken at room temperature for 30 minutes to allow the magnetic beads and antibody to bind sufficiently, after which the mixture was washed three times with 0.1% PBST.

[0095] (3) The magnetic beads were washed once with crosslinking buffer (0.2 M triethanolamine, pH 8.2). Then, 12 mg of DMP was dissolved in 2 mL of crosslinking buffer, added to the magnetic beads, and incubated at room temperature for 1 hour. After the reaction was complete, the crosslinking solution was discarded, and the beads were washed once with 2 mL of blocking buffer (0.1 M ethanolamine, pH 8.2). Then, another 2 mL of blocking buffer was added and the beads were incubated at room temperature for 2 hours. (4) After blocking, the magnetic beads were washed three times with PBS, then washed once with 0.1 M glycine (pH 2.5), and allowed to react for 5 minutes. They were then washed three times with 0.1% PBST, immersed in PBST containing 0.02% NaN3, and stored at 4°C.

[0096] 1.4 In vitro production of Aβo*3F One mg of Aβ42, Aβ40, or other forms of Aβ (Naka Peptide Biochemicals, Inc.) was dissolved in 1 mL of 100% hexafluoroisopropanol (HFIP), vortexed for 5 min, sonicated in a water bath for 10 min, dispensed into EP tubes, allowed to evaporate overnight, and stored at -20°C. Before use, the HFIP-treated Aβ aliquot was dissolved in 50 mM NaOH at a concentration of 1 mg / mL, vortexed for 3–5 min, sonicated for 1 min, and then diluted to 10 μM with pre-chilled PBS. The solution was centrifuged at 21,000 xg for 30–40 min at 4°C, and the precipitate (approximately 5% of the initial volume) was discarded to obtain Aβ monomers. The Aβ monomers were incubated at 25°C for 2 days under static conditions and then incubated overnight at 4°C with protein A magnetic beads cross-linked with 3F antibody. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20-100 mM glycine (pH 2.0) for 3-5 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain sAβo*3F (in vitro produced Aβos).

[0097] 1.5 Isolation and preparation of Aβo*3F in APP / PS1 mouse brain homogenates and CSF of AD patients To prepare Aβo*3F specifically recognized by 3F (Aβo*3F), APP / PS1 mouse brain homogenate and CSF samples from AD patients (derived from the First Affiliated Hospital of Zhengzhou University, with signed informed consent and approval from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University) were incubated overnight at 4 °C with Protein A magnetic beads crosslinked with 3F antibodies. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20 mM to 100 mM glycine (specifically, 20 mM, pH 2.0) for 3 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβo*3F from APP / PS1 mouse brain (mAβo*3F, Aβo*3F isolated from AD mouse brain) or Aβo*3F extracted from AD patient CSF (hAβo*3F, Aβo*3F isolated from AD patient cerebrospinal fluid). The mixtures of Aβ aggregates after 3F immunodepletion, called Aβ-ID, were used as controls: sAβ-ID (produced in vitro), mAβ-ID (produced by isolation from the brains of APP / PS1 mice), and hAβ-ID (produced by isolation from CSF of AD patients).

[0098] Aβ*6E10, a mixture of Aβ aggregates, was prepared by mixing APP / PS1 mouse brain homogenate or AD patient CSF with protein G magnetic beads cross-linked with 6E10 antibody and incubating overnight at 4°C. The mixture was then eluted with 20 mM to 100 mM glycine (specifically, 20 mM, pH 2.0) for 3 to 5 min (specifically, 3 min). This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβ*6E10 from APP / PS1 mouse brain (mAβ*6E10, Aβ*6E10 isolated from AD mouse brain) or Aβ*6E10 extracted from AD patient CSF (hAβ*6E10, Aβ*6E10 isolated from AD patient CSF) (Figure 1). The concentration of Aβ obtained by immunoprecipitation was measured using an Aβ detection kit.

[0099] Example 2 Characterization of the molecular weight and morphology of Aβo*3F 2.1 Experimental materials and methods 2.1.1 Experimental materials Aβ polypeptide: Nakapeptide Biochemical Co., Ltd. Superdex 200 10 / 300 GL molecular sieve: GE Healthcare. A11 antibody: Invitrogen, #AHB0052. Molecular sieve protein marker: GE healthcare. 3–8% Tris-Acetate precast gels: Invitrogen. Copper mesh with carbon support film: Beijing Zhongke Kegyi Co., Ltd. Uranyl acetate: Beijing Zhongkeke Yi Co., Ltd. ECL Chemiluminescence Kit: Pierce Biotechnology. ThT detection reagent: Sigma-Aldrich. Nitrocellulose membrane: Millipore.

[0100] 2.1.2 Experimental equipment AKTA Protein Chromatography: GE Healthcare Life Science, USA. Protein electrophoresis equipment: Bio-rad, USA. Protein membrane transfer system: Bio-rad, USA. HT7700 transmission electron microscope: Hitachi, Japan. Safire2 TM Microplate reader: Tecan Group, Switzerland. Amersham Imager 680 imaging system: GE, USA.

[0101] 2.1.3 Main solutions (1) 20 mM TBS buffer solution: 2.4 g of Tris and 8 g of sodium chloride were weighed and dissolved in deionized water, and the pH was adjusted to 7.5 with dilute hydrochloric acid, and the solution was made up to 1 L. (2) 0.1% TBST: 1 mL of Tween-20 was added to 1 L of TBS and stirred for 20 min. (3) ThT solution: A 100X ThT stock solution (500 μm) was prepared in 50 mM PB (phosphate buffer, pH = 6.5) solution, and diluted to 1X with 50 mM PB (pH = 6.5) before use. (4) 5% skim milk.

[0102] (5) Electrophoresis buffer: 30 g of Tris, 144 g of glycine, and 10 g of SDS were dissolved, and the solution was diluted to 1 L with deionized water and diluted 10 times before use. (6) 5x non-reducing loading buffer (10 mL): 2 mL of 10% SDS, 0.6 mL of 1 M Tris-HCl (pH 6.8), 5 mL of glycerin, and 1 mL of 1% bromophenol blue were taken and made up to 10 mL with deionized water. (7) Membrane transfer buffer: 30 g of Tris and 144 g of glycine were dissolved in deionized water, made up to 1 L, and diluted 10-fold before use.

[0103] 2.2 Preparation and characterization of Aβ monomers, oligomers, and fibrils Aβ monomers were prepared as in 1.4. The Aβ monomers were incubated at 25°C for 0-4 days under static conditions, and the Aβ aggregation state was detected every 24 hours using ThT. The specific procedure is as follows: Aβ samples were diluted to 10 μM with PBS buffer, and then 190 μL of ThT detection solution and 10 μL of test sample were thoroughly mixed in a black ELISA plate to homogenize, and then the plate was subjected to Safire 2 TM Fluorescence was measured using a microplate reader at an excitation wavelength of 440 nm and an emission wavelength of 480 nm. As can be seen from the results, with increasing incubation time, the ThT readings of the Aβ samples gradually increased, indicating the gradual aggregation of Aβ monomers into oligomers and fibrils (Figure 2A).

[0104] 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 h at room temperature. The membranes were incubated with different detection antibodies for 1–2 h at room temperature, then washed three times with 0.1% TBST for 5 min each, and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After three washes with 0.1% TBST for 5 min each, ECL chemiluminescence solution was added to the membranes, and the staining was developed using an Amersham Imager 680 imaging system. Quantitative analysis of blot size and optical density was performed using Image J software. The results show that 6E10 is an antibody that broadly binds to Aβ monomers, oligomers, and fibrils, and can bind to 10 μM and 20 μM Aβ without significant differences in binding. Neither 3F nor A11 antibodies bound to Aβ monomers. Furthermore, they bound to different types of oligomers. The 3F antibody primarily bound to 10 μM Aβos, whereas the A11 antibody primarily bound to 20 μM Aβos. This indicates that the 3F and A11 antibodies recognize oligomers with different conformations (Figure 2B). Furthermore, statistical analysis of the affinity of 3F for 10 μM Aβos obtained after 0–4 days of incubation revealed that the affinity of 3F for Aβos obtained after 2 days of incubation at 10 μM (sAβos) was highest. Therefore, in subsequent experiments, we immunoprecipitated this oligomer (sAβo*3F, in vitro-produced Aβo*3F) with 3F (Figure 2C).

[0105] 2.3 Molecular weight characterization of sAβo*3F First, the molecular weight of sAβo*3F was detected by Western blotting. The specific procedure is as follows: Aβ samples were loaded onto a 15% SDS-PAGE gel or a 3-8% Tris-Acetate gel. For Tris-Acetate gels, samples were loaded onto Novex gels before loading. TMThe mixture was mixed with Tris-glycine sample buffer. Electrophoresis was performed in Tris-glycine electrophoresis buffer (containing 0.5 mM DTT, 1 mM ATP, and 5 mM MgCl2) at 4°C for 4 hours at 150 V. For Western blot analysis, proteins separated by SDS-PAGE or Tris-Acetate gel were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 1–2 hours at room temperature, and then incubated overnight at 4°C with a detection antibody (6E10). The membrane was then washed three times for 5 minutes with 0.1% TBST and incubated with the corresponding secondary antibody (HRP-conjugated goat anti-mouse secondary antibody) for 1 hour 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.

[0106] As can be seen from the SDS-PAGE experiment, sAβo*3F is mainly composed of two types of oligomers with molecular weights of approximately 12 kDa and the other with molecular weights greater than 180 kDa. As can be seen from the native-PAGE results, sAβo*3F is mainly composed of oligomers with molecular weights of approximately 500 kDa. The difference in the electrophoretic results between the two types may be due to the influence of SDS (Figure 2D). To further characterize the molecular weight of sAβo*3F, 500 μL of sAβo*3F or 100 μL of a SEC marker was loaded onto a Superdex 200 10 / 300 GL molecular sieve 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 that the molecular weight of sAβo*3F was 588 kDa (Figure 2E and F).

[0107] 2.4 Molecular weight and morphology characterization of mAβo*3F Aβo*3F was extracted from the brains of APP / PS1 mice (mAβo*3F) 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 over 180 kDa, respectively. However, on native-PAGE, mAβo*3F showed only a single band with a molecular weight of approximately 500 kDa (Figure 2G). SEC analysis revealed that the molecular weight of mAβo*3F was 588 kDa (Figure 2H). Additionally, 10 μL of Aβo*3F was dropped onto a 200-mesh copper mesh and allowed to adsorb for 20 min, dried on filter paper, and negatively stained with 2% uranyl acetate for 30 s. The mesh was then dried on filter paper and air-dried. The samples were examined by a transmission electron microscope (TEM, Hitachi H7700, Japan) at an operating voltage of 120 kV and a magnification of 100,000. As can be seen from the results, mAβo*3F was a particle with a diameter of approximately 10 nm (Figure 2H).

[0108] 2.5 Molecular weight characterization of Aβo*3F in CSF of AD patients The molecular weight and distribution of Aβo*3F (hAβo*3F) extracted from CSF of AD patients were consistent with those of sAβo*3F and mAβo*3F. hAβo*3F showed only one band with a molecular weight of approximately 500 kDa in native-PAGE, but showed two bands with molecular weights of 12 kDa and over 180 kDa in SDS-PAGE (Figure 2, Panel I).

[0109] Example 3: Detection of Aβo*3F neurotoxicity 3.1 Experimental materials N2a cells: National Experimental Cell Resources Sharing Service Platform (NICR). Poly-D-lysine hydrobromide (PDL): Sigma-Aldrich. B27 Supplement (50x): Thermo Fisher Scientific. Neurobasal TM -A medium: Gibco. 70 μm cell nylon screen: BD Bioscience.

[0110] 3.2 Primary neuron culture The brains of C57BL / 6 (purchased from Beijing Huafukang Biotechnology Co., Ltd.) mouse fetuses at 14-15 days of gestation were removed, and the brain membranes were peeled off using HBSS (Hank's Balanced Salt Solution). The hippocampus and cortex were finely chopped with tweezers. The crushed tissue mass was transferred to a 15 mL centrifuge tube and centrifuged at 600 rpm for 3 minutes. The pellet was then washed with 10-fold diluted trypsin digestion solution (containing DNase I). The cells were digested at 37°C for 10 min, gently inverting several times every 5 min. After digestion, the trypsin digestion reaction was stopped with 40 mL of DMEM medium (containing 10% FBS), and the cell suspension was passed through a 70 μm cell strainer to remove undigested tissue clumps. The cells were collected by centrifugation at 1200 rpm for 10 min, resuspended in DMEM medium, and plated onto a 12-well plate. After 2–4 h, the medium was replaced with Neurobasal medium (containing B27, 0.5 mM L-glutamine, 0.5% penicillin and streptomycin), and the culture was continued for 7–9 days before use in subsequent experiments.

[0111] 3.3 Detection of sAβo*3F cytotoxicity This experiment primarily utilized the MTT assay to detect the toxicity of Aβ aggregates to neurons. The specific procedure was as follows: N2a cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells in the culture dish were digested with trypsin, centrifuged, resuspended in medium, and then seeded into a 96-well plate at approximately 5,000 cells per well in 100 μL of medium. After 12 h, the cells were treated with a gradient series of sAβo*3F and sAβos. At the same time, an equal volume of solvent was added to the cells as a control. After 72 h, 25 μL of 5 mg / mL MTT was added to each well. After 3 h 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 wells was used for each sample and control, and each experiment was repeated three times. Cell vitality was calculated by dividing the absorbance (background corrected) of the sample-added wells by the absorbance (background corrected) of the solvent-added wells.

[0112] As can be seen from the MTT results, the 50% inhibitory concentration (IC50) of sAβo*3F on cell viability was approximately 0.186 nM, whereas the IC50 of sAβos before immunoprecipitation was approximately 63.26 nM, a 340-fold difference between the two (Figure 3A and B). Furthermore, when 0.5 nM of sAβo*3F was added to N2a cells, it induced 66% cell death, whereas the same concentrations of sAβos and sAβ-ID had no significant neurotoxic effects. When sAβos reached 200 nM, it induced 70% cytotoxicity, whereas the neurotoxicity of sAβ-ID at this time was only 32% (Figure 3C). These results demonstrate that sAβo*3F is a potent oligomer with neurotoxicity.

[0113] 3.4 Detection of mAβo*3F cytotoxicity The IC50 of mAβo*3F for N2a cells was approximately 0.111 nM, as detected by the MTT assay, and the IC50 for primary neuronal cells was approximately 0.057 nM (Figure 3D). Specifically, 0.5 nM of mAβo*3F was able to induce 78% cell death in primary neurons, whereas the same concentrations of mAβ*6E10 and mAβ-ID had no obvious cytotoxicity. When the concentration was increased to 200 nM, mAβ*6E10 was able to induce 54% neurotoxicity, whereas the neurotoxicity of mAβ-ID was only 20% (Figure 3E).

[0114] 3.5 Detection of cytotoxicity of hAβo*3F The IC50 of hAβo*3F on primary neurons was approximately 0.076 nM as detected by the MTT assay (Figure 3F). Specifically, when 0.2 nM of hAβo*3F was added to primary neurons, it was able to reduce cell vitality by 68%, whereas 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 vitality by 66%, whereas hAβ-ID reduced cell vitality by only 40% (Figure 3G).

[0115] Example 4 Effect of Aβo*3F on the expression levels of cytokines in glial cells 4.1 Experimental materials and methods 4.1.1 Experimental materials BV2 cells: NICR. Reverse transcription kit: Kangwei Century Co., Ltd. UltraSYBR Mixture (Low ROX): Kangwei Century Co., Ltd. 4.1.2 Experimental equipment 7500 Fast real-time quantitative PCR instrument: Applied Biosystems. T100 Thermal Cycler PCR Instrument: BIO-RAD. NanoDrop Microvolume Spectrophotometer: Quawell.

[0116] 4.2 Aβo*3F can increase the expression levels of inflammatory cytokines in microglia. BV2 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells in the culture dish were digested with trypsin, centrifuged, resuspended in medium, and then seeded into a 12-well plate at approximately 5 x 105 cells / mL per well. After 12 h, BV2 cells were treated with different Aβ samples; at the same time, the same volume of solvent was added to the cells as a control. After 48 h, intracellular proinflammatory cytokine expression levels were detected by fluorescent quantitative PCR (qPCR). The specific procedures were as follows: Total cellular mRNA was prepared using standard methods, and the mRNA concentration was measured using a microvolume spectrophotometer. After reverse transcription to obtain cDNA according to the reverse transcription kit's instructions, the expression level of the target gene was detected using EasyQuick RT MasterMix. The primers used were as follows:

[0117] mTNF-α:5'-GATTATGGCTCAGGGTCCAA-3' (SEQ ID NO:1), 5'-GCTCCAGTGAATTCGGAAAG-3(SEQ ID NO:2); mIL-1β:5'-CCCAAGCAATACCCAAAGAA-3' (SEQ ID NO:3), 5'-GCTTGTGCTCTGCTTGTGAG-3'(SEQ ID NO:4); mIL-6:5'-CCGGAGAGGAGACTTCACAG-3' (SEQ ID NO:5), 5'-TTGCCATTGCACAACTCTTT-3'(SEQ ID NO:6); GAPDH:5'-TGAATACGGCTACAGCAACA-3'(SEQ ID NO:7), 5'-AGGCCCCTCCTGTTATTATG-3' (SEQ ID NO:8).

[0118] As can be seen from the qPCR results, 0.3 nM mAβo*3F, 200 nM mAβ*6E10, and 200 nM mAβ-ID were able to induce a significant increase in the expression levels of microglial inflammatory cytokines, including TNF-α, IL-6, and IL-1β, while 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID showed no obvious stimulatory effect (Figure 4A).

[0119] 4.3 Primary astrocyte culture One- to two-day-old C57BL / 6 mouse neonates were taken and disinfected by immersion in 75% alcohol. The cerebrum was then removed and immersed in HBSS buffer. The meninges were peeled off and the cerebrum was cut into small pieces with tissue scissors. The pieces were placed in 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. The digestion was continued for 5 minutes. The cells were gently inverted several times every 1 min. After digestion, the trypsin digestion reaction was stopped with 40 mL of DMEM medium (containing 10% FBS), and the cell suspension was passed through a 70 μm cell strainer to remove undigested tissue clumps. The cells were collected by centrifugation at 1200 rpm for 10 min, resuspended in DMEM medium, plated into T-75 culture bottles, and cultured at 37°C in DMEM medium for 5–7 days. The medium was changed every 2–3 days. After 7 days of culture, the cells were digested with trypsin and plated into 12-well plates for use.

[0120] 4.4 Aβo*3F can stimulate the expression of inflammatory cytokines in astrocytes. Primary astrocytes were treated with different Aβ samples and their effects on the expression levels of inflammatory cytokines in primary astrocytes were examined by qPCR. The specific procedure was the same as in 4.2. The primers used were as follows:

[0121] miNos:5'-CACCTGGAACAGCACTCTCT-3'(SEQ ID NO:9), 5'-CTTTGTGCGAAGTGTCAGTG-3'(SEQ ID NO:10); mTNF-α: SEQ ID NO: 1 and 2; mIL-1β: SEQ ID NO: 3 and 4; mIL-6: SEQ ID NO: 5 and 6; GAPDH: SEQ ID NO:7 and 8.

[0122] As can be seen from the results, 0.3 nM mAβo*3F, 200 nM mAβ*6E10, and 200 nM mAβ-ID could significantly enhance the expression levels of inflammatory cytokines, including TNF-α, IL-6, IL-1β, and iNos, in primary astrocytes, whereas 0.3 nM mAβ*6E10 or 0.3 nM mAβ-ID had no obvious effect on the expression levels of inflammatory cytokines in astrocytes (Figure 4B).

[0123] 4.5 Aβo*3F disrupts astrocyte-mediated synaptogenesis. Astrocytes can induce synapse formation by secreting synaptic factors such as TSP1 and Gpc4 / 6. mAβo*3F was added to primary astrocytes, and changes in the expression levels of several synaptic factors in astrocytes were detected by qPCR. The primers used were as follows:

[0124] mGpc4:5'-CTGGAGGGTCCTTTCAACATT-3' (SEQ ID NO:11), 5'-GACATCAGTAACCAGTCGGTC-3'(SEQ ID NO:12); mGpc6:5'-TAGTCCTGTATTGGCAGCCAC-3' (SEQ ID NO:13), 5'-GGCTAATGTCTATAGCAGGGAA-3'(SEQ ID NO:14); mTSP1:5'-GGTAGCTGGAAATGTGGTGCGT-3' (SEQ ID NO:15), 5'-GCACCGATGTTCTCCGTTGTGA-3'(SEQ ID NO:16); GAPDH: SEQ ID NO:7 and 8.

[0125] As can be seen from the results, treatment of cells 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 obvious effect. Only increasing the concentration of mAβ-ID up to 200 nM was able to reduce the expression level of Gpc4 in astrocytes (Figure 4C).

[0126] Example 5: Effects of Aβo*3F on cognition in mice and damaging effects on neurons in the brain 5.1 Experimental materials and methods 5.1.1 Experimental materials Rapid Golgi staining kit: FD NeuroTechnologies. Ziehl-Neelsen staining kit: Solarbio.

[0127] 5.1.2 Experimental equipment Brain stereotactic injection system: Chinese Academy of Medical Sciences. Y-maze equipment: Chinese Academy of Medical Sciences. Novel object recognition device: Chinese Academy of Medical Sciences.

[0128] 5.2 Experimental animals Three-month-old C57BL / 6 mice were housed in a clean room at 22±2°C and 45%±10% humidity, with free access to food and water and a 12-hour light / 12-hour dark cycle. All animal experiments were performed in accordance with the "Guidelines for the Care and Use of Laboratory Animals of the China Public Health Service." All mouse experiments were approved by the Tsinghua University Animal Care and Use Committee.

[0129] 5.3 Stereotactic injection into the brain Three-month-old C57BL / 6 mice were randomly divided into six groups of 6–8 mice each: one group injected with 2.5 nM (45.5 pg) mAβo*3F, one group injected with 2.5 nM (45.5 pg) mAβ*6E10, one group injected with 2.5 nM (45.5 pg) mAβ-ID, one group injected with 600 nM (10.9 ng) mAβ*6E10, one group injected with 600 nM (10.9 ng) mAβ-ID, and a control group injected with Tris-Gly vehicle. Mice were deeply anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg) and then secured to a stereotaxic brain injection platform. The skin over the brain was incised along the midline to expose the skull. The injection site was positioned using a stereotaxic instrument at AP = -1.7 mm, ML = ±1.0 mm, and DV = -1.5 mm from the bregma. After a craniotomy along the stereotaxic point was performed with a skull drill, 4 μL of solution was injected into each half of the brain bilaterally at a rate of 0.4 mL / min. After the injection, the needle was left in place for 3 min to allow complete absorption of the sample. The surgical site was irrigated with sterile saline and the incision was sutured. After surgery, the mice were monitored and provided with postoperative care. 24 h after stereotaxic injection, behavioral and cognitive performance were assessed, and the mice were then dissected for physiological and biochemical analyses.

[0130] 5.4 Aβo*3F severely impairs memory ability in mice Novel object recognition experiments and Y-maze experiments are used to detect changes in cognitive abilities of mice.

[0131] 5.4.1 Novel object recognition experiment The novel object recognition experiment was designed based on the spontaneous tendency of mice to show more interaction with unfamiliar novel objects. The experiment was divided into three main stages. (1) During the familiarization phase, a white box measuring 40 cm wide x 40 cm deep x 40 cm high was prepared. When there was no object in the box, each mouse was allowed to freely explore the box for 5 min. After 1 h, the mice were injected with Aβ samples. (2) In the training phase, after 24 h, two identical objects were placed in the box, and each mouse was placed in the same box as in the first phase. Each mouse was allowed to freely explore the box for 5 min. The number of times the mouse sniffed or touched each object was recorded.

[0132] (3) In the test phase, after 6 hours of training, the object on the right side was replaced with a new object that was different in material, color, and shape. Each mouse was allowed to explore freely for 5 minutes, and the number of times the mouse smelled and touched the two objects was recorded to detect the mouse's memory ability. The recognition index was calculated using the formula: (number of times for new object - number of times for old object) / (number of times for new object + number of times for old object). As can be seen from the results, the recognition index for novel objects of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group, whereas the recognition index for novel objects of mice in other Aβ-treated groups showed no significant difference compared to the control group, indicating that Aβo*3F significantly reduced the memory ability of mice (Figure 5A).

[0133] 5.4.2 Y-maze experiment After the novel object recognition experiment, the spatial memory ability of the mice was detected by a Y-maze experiment. The Y-maze consisted of three identical arms, with a 120-degree angle between each arm. The dimensions of each arm were 8 cm x 30 cm x 15 cm (width x length x height). The experiment was mainly divided into two stages. (1) In the training phase, three arms were randomly named A, B, and C. Arms A and B were open throughout the experimental phase, while arm C was closed as a new arm during the training phase. After each mouse entered arm A, it was allowed to freely explore arms A and B for 10 min. (2) After 1 h of training, the C arm was opened, and the mice were placed in the A arm again, one after the other. The mice were allowed to explore the A, B, and C arms for 5 min, and the time spent in the three arms was analyzed. All testing procedures were recorded by a camera installed above the Y-maze.

[0134] As can be seen from the results, the duration in the novel arm of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group. In contrast, there was no significant difference in the duration of the novel arm in mice injected with 2.5 nM mAβ*6E10, 2.5 nM mAβ*ID, or 600 nM mAβ*ID compared to the control group. mAβo*3F, but not mAβ*6E10 or mAβ-ID, was able to induce severe memory impairment in mice at low concentrations, whereas mAβ*6E10 was only able to induce similar memory impairment at concentrations up to 600 nM (Figure 5B).

[0135] 5.5 Aβo*3F significantly reduces dendritic spine density in mouse neurons Mouse brain samples were stained using the FD Rapid Golgi Stain Kit. 24 h before dissection, solutions A and B in the kit were mixed 1:1 and stored in a dark place. After sacrifice, the brain was removed without perfusion and divided along the midline into left and right hemispheres. Blood on the surface of the left hemisphere was washed with PBS, and the solution on the surface of the tissue was removed with filter paper. The tissue was then placed in the pre-prepared A+B solution. The solution was changed once after 24 h and left at room temperature for two weeks, protected from light. For best results, the tissue was gently inverted twice a week during the immersion period. After two weeks, the tissue was transferred to solution C, the solution was changed once after 24 h, and left at room temperature for five days, protected from light. Then, 100 μm-thick frozen sections were obtained using a freezing microtome at -22°C. The sections were allowed to air dry at room temperature before staining began. First, sections were washed twice with Milli-Q water for 4 min each. Then, a working staining solution was prepared. The sections were placed in the working staining solution for 15 min and then washed twice with Milli-Q water for 5 min each. After staining, sections were dehydrated in 50%, 75%, 95%, and 100% ethanol, each for 4 min. The sections were then placed in xylene, sealed with neutral resin, and observed. Dendrites and dendritic spines of neurons in the hippocampal CA1 region were observed using a 100x objective lens on an Olympus inverted microscope. Dendritic spine density was determined using Image J software analysis (dendritic density = number of dendritic spines / dendritic length).

[0136] We calculated the dendritic spine density and found that the dendritic spine density of neurons in the CA1 region of the brains of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group, whereas 2.5 nM mAβ*6E10, 2.5 nM mAβ-ID, or 600 nM mAβ-ID had no obvious effect on the dendritic spine density of neurons in the brains of mice (Figure 5C, D).

[0137] 5.5 Aβo*3F significantly reduces the number of hippocampal neurons in mice 5.5.1 Paraffin embedding and sectioning After the behavioral experiments, mice were deeply anesthetized with pentobarbital sodium (50 mg / kg) and intracardially injected with ice-cold PBS containing heparin (10 U / mL). The mice were then sacrificed and dissected. The cerebrum was removed and separated into left and right hemispheres along the midline. The left hemisphere was immersed in 4% paraformaldehyde for 48 hours, then dehydrated in gradients of 50%, 70%, 80%, and 90% ethanol for 1 hour each. The tissue was then dehydrated once in 100% ethanol for 1 hour, followed by 50% xylene for 30 minutes. After dehydration, the tissue was embedded in paraffin for 4 hours. 5 μm tissue sections were obtained using a paraffin tissue slicer.

[0138] 5.5.1 Ziehl-Neelsen staining Five-micrometer paraffin sections were heated at 60°C for 30 minutes, then dewaxed by immersion in 100% xylene, 50% xylene, and 100% alcohol for 10 minutes each, followed by immersion in 75% ethanol, 50% ethanol, 30% ethanol, and deionized water for 5 minutes each. Following the instructions for Ziehl-Neelsen staining, sections were stained with cresol purple solution and heated at 56°C for 1 hour. After differentiation at room temperature for 1–3 minutes until the background was nearly colorless, sections were destained in graded alcohols (70%, 95%, and 100%; 20 seconds each). The sections were then immersed in xylene and sealed with neutral resin. Data were then collected and analyzed microscopically. As can be seen from the results, 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly reduced the number of neurons in the CA1 and DG regions of the mouse brain, whereas 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 5E, F, G).

[0139] Example 6 Aβo*3F activates glial cells and induces neuroinflammation in the mouse brain 6.1 Experimental materials and methods 6.1.1 Experimental materials Anti-GFAP antibody: Cell Signaling Technology, #3670. Anti-Iba-1 antibody: GeneTex, #GTX101495. BCA kit: Thermo Fisher Scientific. RIPA strong lysis solution: Solarbio. Protease inhibitors: Millipore. Phosphatase inhibitor (100x): Solarbio. Mouse IL-1β detection kit: Biolegend. Mouse IL-6 detection kit: Biolegend.

[0140] 6.1.2 Experimental equipment MD-M5 microplate reader: Molecular Devices, USA. Tissue Lyser II tissue disrupter: Qiagen.

[0141] 6.1.3 Immunohistochemistry After dewaxing, 5 μm paraffin sections were immersed in citrate antigen repair solution (3 g trisodium citrate and 0.4 g citric acid dissolved in deionized water and made up to 1 L), boiled in a water bath for 15 min, and then allowed to cool at room temperature. After antigen repair, the sections were washed three times with PBS for 5 min each, and then fixed in 80% (vol / vol) methanol containing 0.3% HO to remove endogenous catalase. After three 5-min washes with PBS, the sections were placed in 0.3% Triton X-100 permeabilization solution for 20 min at room temperature, and then washed three times with PBS for 5 min each. The sections were blocked with 10% donkey serum at room temperature for 1 h, then incubated overnight at 4°C with the corresponding primary antibody. On the second day, the sections were washed three times with PBS for 5 min each, and then the corresponding secondary antibody was added and incubated at room temperature for 1 h. After washing three times with PBS, the sections were developed and observed.

[0142] 6.2 Aβo*3F can activate microglia and astrocytes We stained for Iba-1, a marker of hippocampal microglia, and found that 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly activated microglia in the DG and CA1 regions of the hippocampus, increasing Iba-1 expression levels in microglia, but had no significant effect on mice treated with 2.5 nM mAβ*6E10 or mAβ-ID (Figure 6A, C, D). Furthermore, 2.5 nM mAβo*3F and 600 nM mAβ*6E10 significantly induced microglia in the DG region to adopt an amoeboid state, characterized by enlarged cell bodies and reduced branching, indicating that microglia were in an overactivated state (Figure 6A). We stained for GFAP, a marker of astrocytes in the mouse hippocampus, and found that intracerebral injection of 2.5 nM mAβo*3F and 600 nM mAβ*6E10 activated astrocytes in the hippocampal DG and CA1 regions and increased the expression level of hippocampal GFAP (Figure 6, B, E, F).

[0143] 6.3 Aβo*3F increases the levels of inflammatory cytokines in the hippocampus of mouse brains The levels of inflammatory cytokines (IL-6 and IL-1β) in mouse brains were detected using IL-6 and IL-1β ELISA kits, respectively, according to the manufacturer's instructions. Briefly, brain homogenates were diluted to the specified fold and added to ELISA plates. The plates were pre-coated with the corresponding capture antibodies, followed by the addition of the corresponding detection and secondary antibodies. TMB was used as the substrate, and the absorbance was measured at 450 nm using an MD-M5 microplate reader. 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, whereas the brain levels of mice injected with 2.5 nM mAβ*6E10 or 2.5 nM mAβ-ID were not significantly different from those of the control group (Figure 6G, H).

[0144] Example 7 Detection of Aβo*3F levels in CSF and plasma of AD patients (MSD method) 7.1 Experimental materials and methods 7.1.1 Experimental materials Aβ Multifactorial Detection Kit (4G8): Meso Scale Diagnostics.

[0145] 7.1.2 Experimental equipment MSD-S600 electrochemiluminescence detection instrument: Meso Scale Diagnostics, USA.

[0146] 7.2 Detection of Aβo*3F levels in plasma and CSF of AD patients To evaluate the correlation between Aβos (Aβo*3F), which is specifically recognized by 3F, and the pathogenesis of AD patients, we measured the levels of Aβ42 and Aβ40 contained in Aβo*3F isolated from CSF and plasma of AD patients. Both the human plasma and CSF samples used in this study were approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University, and all subjects signed 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 mild cognitive impairment (MCI) patients with a mean age of 73.3 years (age range 57-84 years), 20 AD patients with a mean age of 69.4 years (age range 52-85 years), and 20 healthy subjects with a mean age of 70.9 years (age range 65-78 years). Additionally, CSF samples were collected from seven AD patients with a mean age of 68 years (age range 59-77 years) and seven non-demented subjects with a mean age of 66.3 years (age range 48-79 years).

[0147] To extract Aβo*3F from plasma and CSF of AD patients and healthy elderly subjects, human plasma or CSF samples were first added to 100 μL of Protein A / G-agarose gel and incubated at 4°C for 1 h to remove endogenous IgG. The sample and the Protein A magnetic beads crosslinked with 3F antibodies were then incubated at room temperature for 2 h at 4°C. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20 mM glycine (pH 2.0) for 3 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris. The concentration of Aβo*3F extracted by immunoprecipitation was detected using the MSD Aβ Multifactor Detection Kit (4G8). The results show that the levels of Aβ42 and Aβ40 contained in Aβo*3F in CSF and plasma of AD patients were significantly increased compared to healthy elderly subjects (Figure 7). Furthermore, the levels of Aβ42 and Aβ40 contained in Aβo*3F in the plasma of MCI patients were significantly higher than those of controls, and these levels gradually increased with the progression of AD pathology (Figure 7C and 7D). These results suggest that Aβo*3F levels in the CSF and plasma of AD patients and AD-derived MCI patients were significantly higher than those of healthy elderly individuals and closely correlated with the progression of AD pathology. Therefore, Aβo*3F could be used as a biomarker for AD diagnosis, especially for early diagnosis. Statistics were performed using GraphPad Prism 8 software, combining one-way ANOVA and Tukey's multiple comparison test. 95% confidence intervals were used. * indicates a statistically significant difference (P<0.05). ** indicates a highly statistically significant difference (P<0.01). *** indicates a highly statistically significant difference (P<0.001). **** indicates P<0.0001, indicating a highly statistically significant difference.

[0148] Example 8: A vaccine (ABW vaccine) produced using Aβo*3F as an immunogen significantly improves cognitive function in AD mice and reduces pathological changes in the brains of mice. 8.1 Immunization of mice and measurement of antibody titers Female BalB / c mice aged 4-6 weeks were divided into groups of six. Aβo*3F and Alum adjuvant (ThermoFisher, 77161) were mixed at an Alum adjuvant to antigen ratio of 1:1 to 1:3 (v / v). Each mouse was immunized with 100 μg of Aβo*3F subcutaneously every two weeks. Blood was collected from the vein before immunization, and serum was collected and stored at -80°C. Blood was collected from the vein two weeks after the third and fourth immunizations, and serum was collected. Antibody titers in the serum were detected by ELISA.

[0149] A 96-well plate was coated with Aβo*3F (0.5 μg / 100 μL) overnight at 4°C. After coating, the coating solution was shaken off and the plate was washed three times with 0.05% PBST. 300 μL of 3% BSA was added per well and blocked at 37°C for 2 hours. The plate was washed twice with 0.05% PBST. Serum (diluted with 3% BSA) was added at 100 μL per well and incubated at 37°C for 2 hours. The plate was washed eight times with 0.05% PBST. HRP-conjugated anti-IgG secondary antibody was added at 100 μL per well and incubated at 37°C for 1 hour. The plate was washed six times with PBST. 100 μL of TMB substrate coloring solution was added per well and allowed to develop at room temperature for 10 minutes. The reaction was stopped by adding 100 μL of 0.1 M H2SO4 per well. The absorbance value at 450 nm was detected using a microplate reader. The results showed that after three immunizations, the antibody titer of anti-Aβo*3F was significantly improved, demonstrating that Aβo*3F was able to produce anti-Aβo*3F-specific antibodies through immune stimulation (Fig. 8).

[0150] 8.2 ABW vaccine can significantly improve the cognitive ability of AD mice and alleviate pathological changes in the brain of mice 8.2.1 Water maze experiment The water maze consisted of a 110 cm diameter pool and a 10 cm diameter platform. The pool contained opaque water (22 ± 1°C). Each pool was marked with a different symbol to help mice locate and identify the platform. During the 5-day training period, the platform was located 1 cm below the water surface. Mice were randomly placed in the pool and allowed to swim for 60 s to find the platform, after which they remained on the platform for 10 s. If the mouse found the platform, the experiment was terminated and the latency to find the platform was recorded. Mice that could not find the platform were guided to the platform and the latency period was recorded as 60 s. After each training session, the mice were wiped with a dry towel, dried using a heater, and placed in their cages. Training was conducted twice daily, with a 3-4 h interval between sessions. The latency period, swimming distance, average swimming speed, and exploration mode of each group of mice were recorded. Twenty-four hours after the final learning test, the platform was removed, and a randomly selected entry point was placed in the water. The mice were then allowed to swim for 60 seconds. The memory retention of the mice was measured in the absence of the platform. The maze was monitored and recorded using a camera mounted above the maze, and parameters such as time spent in the target quadrant, swimming distance, average swimming speed, search path, and number of passes were analyzed and measured using software.

[0151] Active immunotherapy was performed on 6-month-old AD transgenic mice (APP / PS1) by injecting the vaccine. Two weeks after the fourth immunization, behavioral analysis was performed. Water maze experiments demonstrated that the vaccine improved the spatial cognitive abilities of AD transgenic mice (Figure 9). During the training period, the time it took for ABW vaccine-treated AD mice to reach the platform was significantly shorter than that of PBS control AD mice (Figure 9A). In a platform removal test, ABW vaccine-treated AD mice demonstrated clear spatially directional swimming behavior, requiring significantly less time to reach the platform (Figure 9B), making more passes (Figure 9C), and spending longer time in the target quadrant (Figure 9D) than PBS control AD mice.

[0152] 8.2.2 Measurement of Aβ levels in brain homogenates Preparation of brain homogenate: Mouse right brains were homogenized in RIPA buffer containing protease inhibitors. The tissue was then centrifuged at 14,000 g for 30 min at 4°C, and the supernatant containing soluble Aβ (RIPA-soluble fraction) was collected. The insoluble precipitate was resuspended in guanidine hydrochloride buffer (5.0 M guanidine hydrochloride, pH 8.0) and centrifuged at 14,000 g for 1 h at 4°C to obtain the supernatant containing insoluble Aβ (guanidine-soluble fraction). Aβ40 and Aβ42 were detected using IBL's Aβ40 ELISA and Aβ42 ELISA kits. The results revealed that ABW vaccine treatment could significantly reduce the levels of soluble and insoluble Aβ40 and Aβ42 in the brains of AD mice (Fig. 10A-C).

[0153] 8.2.3 Immunostaining of Aβ in mouse brain To examine the effect of ABW vaccine treatment on Aβ plaques in the brains of AD mice, we used immunohistochemistry to detect the levels of 6E10-positive Aβ plaques in the brains of AD mice. The results showed that the stained area of Aβ plaques in the brains of mice treated with ABW vaccine was significantly reduced compared to the PBS control group (Figure 11).

[0154] Example 9: Single-chain antibody 7B, which specifically binds to Aβo*3F and was obtained based on phage screening technology, significantly improves the cognitive ability of AD mice and alleviates pathological changes in the brain of mice. 9.1 Screening and affinity measurement of single-chain antibody 7B that specifically binds to Aβo*3F obtained based on phage screening technology.

[0155] This invention is based on the phage screening technology described in Patent CN101463082A. Aβo*3F was diluted to 10-100 μg / mL in coating buffer (PBS, pH 7.4), and 4 mL of the solution was added to an immunotube and coated overnight at 4°C. After four rounds of enrichment and screening, a positive clone was obtained and identified by sequencing. The single-chain antibody sequence is shown in SEQ ID NO:24 (the light chain CDR sequence and heavy chain CDR sequence are shown in SEQ ID NO:25-30, respectively) and was named 7B. ELISA analysis showed that this antibody had high affinity for Aβo*3F (Figure 12).

[0156] 9.2 Antibody 7B can significantly improve the cognitive ability of AD mice and alleviate pathological changes in the brain of mice. In this study, APP / PS1 transgenic mice were intranasally administered with either 7B antibody or PBS solvent control once daily, and cognitive ability was assessed by a novel object recognition test 21 days later. The level of senile plaques in the brains of APP / PS1 transgenic mice was assessed by thioflavin S (THS) staining.

[0157] The method for the mouse novel object recognition experiment was as described in 5.4.1. The results showed that the cognitive ability of APP / PS1 mice in the PBS solvent control group to recognize the novel object was lower than that of the WT group, while the cognitive ability of APP / PS1 mice in the 7B antibody treatment group was significantly improved compared to that of the PBS group (Figure 13). Staining of mouse brain sections with thioflavin S (THS) revealed that 7B antibody treatment could significantly reduce the levels of senile plaques in the cortex and hippocampus of APP / PS1 transgenic mice (Figure 14).

[0158] Example 10 Binding of optimized 3F antibodies to Aβo*3F oligomers The 100th amino acid, K, of the 3F antibody was mutated to R (if the MA residue introduced for expression as a single-chain antibody is not taken into account, it is considered to be the 98th amino acid in the antibody numbering; this mutant was designated the K98R mutant, and its sequence is shown in SEQ ID NO: 31). The binding of the K98R mutant to Aβo*3F oligomers was examined. The method of use was as follows.

[0159] The above Aβ oligomers were coated onto a 96-well plate at 100 ng / well and left overnight at 4°C. The next day, the plate was blocked with 3% BSA for 2 hours at room temperature, washed twice with PBST, and then dried by tapping. The K98R mutant and 3F were diluted 14 times starting from 1 μg / mL. The diluted antibody solution was added to the Aβ oligomer-coated ELISA plate in multiple wells, incubated for 1 hour at 37°C, washed three times with PBST, and dried by tapping. Goat anti-human IgG-HRP diluted 1:10,000 was added, incubated for 45 minutes at 37°C, washed three times with PBST, and dried by tapping. TMB color development solution was added for approximately 15 minutes, and the plate was stopped with stop solution. Data were read at OD450nm, analyzed, compared, and plotted using GraphPad.

[0160] The results are shown in Figure 15. The experiment demonstrated that the binding affinity of the K98R mutant to Aβo*3F oligomers was further improved, suggesting that the combination of this mutant and Aβo*3F oligomers may be more effective in treating neurodegenerative diseases. The inventors investigated the cause of this result and found that the 98th amino acid, when numbered according to IMGT, is also a residue in the heavy chain CDR3 of the 3F antibody (when numbered according to IMGT, the heavy and light chain CDR sequences of the 3F antibody are CDR-H1:GFTFSSYA (SEQ ID NO:32), CDR-H2:ISNLGLTT (SEQ ID NO:33), CDR-H3:AKTTSRFDY (SEQ ID NO:34), CDR-L1:QSISSY (SEQ ID NO:35), CDR-L2:KAS, and CDR-L3:QNSAVRPVT (SEQ ID NO:36)). The present inventors simultaneously performed the K98R mutation on the sequences of several other mutants, and obtained similar results (data not shown).

[0161] Equivalent Scheme While various embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of numerous other means and / or structures for achieving the functions described herein and / or obtaining one or more of the results and / or advantages described herein, and all such variations and / or modifications are intended to be included within the scope of the present invention. More broadly, those skilled in the art will readily appreciate that all parameters, materials, and settings described herein are exemplary, and that the actual parameters, materials, and / or settings will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalent schemes to the specific embodiments of the present invention described herein. Thus, it should be understood that the foregoing embodiments and examples are presented merely by way of example, and that the invention may be practiced otherwise than as specifically described and claimed, within the scope of the appended claims and their equivalents. Any combination of two or more such features, systems, articles, materials, and / or methods, if not mutually inconsistent, is within the scope of the present invention.

[0162] The term "and / or," as used in the present specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjoined in some cases but not in other cases. In addition to the elements specifically identified by the term "and / or," other elements may optionally be present, whether associated with those specifically identified elements or not, unless expressly indicated otherwise. Thus, as a non-limiting example, when used in conjunction with an open term such as "comprising," a reference to "A and / or B" can, in one embodiment, refer to A without B (optionally including elements other than B), in another embodiment, refer to B without A (optionally including elements other than A), in yet another embodiment, include both A and B (optionally including other elements), and so forth.

[0163] As used herein and in the 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., the inclusion of at least one of a plurality of elements or a list of elements, but including more than one, including a plurality of elements and optionally other items not listed. When used in the claims, terms clearly indicating the contrary, such as "only one of" or "exactly one of," "consisting of" refers to the inclusion of exactly one element of a plurality of elements or a list of elements. Generally, as used herein, the term "or" should only be understood as indicating an exclusive alternative scheme (i.e., "one or the other, but not both") when preceded by terms of exclusion, such as "either," "one of," "only one of," or "exactly one." "Consisting essentially of" when used in the claims should have its ordinary meaning in the field of patent law.

[0164] As used herein and in the claims, when referring to a list of one or more elements, the term "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 not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the term "at least one" refers, may optionally be present, whether or not related to those specifically identified elements. Thus, as a non-limiting example, "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") can mean, in one embodiment, at least one, optionally more than one, A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally more than one, B, and no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements); etc.

[0165] In the claims and the above specification, all conjunctions such as "comprising," "including," "with," "comprises," "containing," "relating to," "having," etc. are understood to be open, i.e., to mean including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively.

[0166] The use of sequence terms in the claims, such as "first," "second," "third," etc., to modify the claim elements themselves does not imply any priority, precedence, or order of one claim element relative to another claim element, or the chronological order in which actions are performed in a method, but is used solely as a tag (but used in numerical terms) to distinguish one claim element with a certain name from another element with the same name to distinguish the claim elements.

Claims

1. 1. An isolated Aβo*3F comprising: the Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody or a variant thereof, and has a total molecular weight of approximately 588 kDa based on analysis by size exclusion chromatography (SEC); The light chain CDR sequences and heavy chain CDR sequences of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively; The mutant is the K98R mutant or 7B antibody, whose light chain CDR sequence and heavy chain CDR sequence are shown in SEQ ID NO: 25-30 or SEQ ID NO: 32-35 and KAS, respectively. Aβo*3F.

2. It is produced in vitro Aβo*3F according to claim 1.

3. A composition comprising the Aβo*3F of claim 1 or 2.

4. an immunogenic composition, a pharmaceutical composition or a vaccine The composition of claim 3.

5. Further comprising a pharmaceutically acceptable excipient and / or adjuvant The composition according to claim 3 or 4.

6. The composition may further comprise another active ingredient, and preferably the other active ingredient is other forms of Aβ oligomers, protofibrils, and mature fibrils, such as Aβ dimers, trimers, hexamers, dodecamers, amylospheroids (ASPDs), amyloid beta-derived diffusible ligands (ADDLs), etc.; preferably, the other active ingredient is a binding agent that specifically binds to Aβo*3F; preferably, the binding agent that specifically binds to Aβo*3F is 3F, K98R, and 7B; more preferably, the binding agent that specifically binds to Aβo*3F and the Aβo*3F are in separate states for simultaneous or sequential administration. The composition according to any one of claims 3 to 5.

7. Dissolving Aβ42, Aβ40 or other forms of Aβ in 50 mM NaOH at a concentration of 1 mg / mL, vortexing for 3-5 minutes, sonicating for 1 minute, and then diluting to 10 μM with pre-chilled PBS; Centrifuging at 21,000 g at 4°C for 30 to 40 minutes, discarding the precipitate (approximately 5% of the initial volume), to obtain Aβ monomers; Incubating the Aβ monomers at 25°C for 2 days under static conditions, and then incubating them with 3F antibody-crosslinked Protein A magnetic beads at 4°C overnight; On the second day, the magnetic beads are washed three times with 0.1% PBST, and then eluted with 20-100 mM glycine (pH 2.0) for 3-5 minutes, and the elution is repeated twice, followed by neutralizing the eluate to pH 7 with 1 M Tris to obtain Aβo*3F. A method for producing Aβo*3F according to claim 1 or 2 in vitro.

8. 1. Use of Aβo*3F as a target in the manufacture of a medicament for preventing and / or treating MCI and / or AD in a subject, The Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody or a variant thereof, and has a total molecular weight of approximately 588 kDa based on analysis by size exclusion chromatography (SEC). The light chain CDR sequence and the heavy chain CDR sequence of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively. The variant is the K98R variant or the 7B antibody, and the light chain CDR sequence and the heavy chain CDR sequence are shown in SEQ ID NOs: 25-30 or SEQ ID NOs: 32-35 and KAS, respectively. Use of Aβo*3F.

9. The drug is an antiserum, an antibody, or a small molecule drug that specifically binds to Aβo*3F, and preferably, the antibody is a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof.

9. The use according to claim 8.

10. 3. The binding agent that specifically binds to Aβo*3F according to claim 1 or 2, wherein the binding agent is 3F, K98R and 7B. Binder.

11. The binding agent is an antiserum, an antibody, or a small molecule drug that specifically binds to Aβo*3F, and preferably, the antibody is a polyclonal antibody or a monoclonal antibody or an antigen-binding fragment thereof. The binder of claim 10.

12. The method includes a step of immunizing an animal with an immunizing effective amount of Aβo*3F according to claim 1 or 2, or screening a phage-displayed peptide library, an antibody library, or a compound library using the Aβo*3F according to claim 1 or 2 as a substrate. A method for producing the binder of claim 10 or 11.

13. The animals are mice, rats, guinea pigs, rabbits, sheep, goats, monkeys, horses, cows, alpacas, and non-human primates. The method of claim 12.

14. The present invention relates to a method for administering a medicament for the treatment of a disease comprising administering a medicament for a patient with ... composition.

15. Use of Aβo*3F according to claim 1 or 2, a composition according to any one of claims 3 to 6, 14 and 15, or a binding agent according to claim 10 or 11 in the manufacture of a medicament for preventing and / or treating MCI and / or AD in a subject.

16. Used as a drug to prevent and / or treat MCI and / or AD in a subject; Aβo*3F according to claim 1 or 2, a composition according to any one of claims 3 to 6, 14 and 15, or a binding agent according to claim 10 or 11.

17. 1. A method for preventing and / or treating MCI and / or AD in a subject, comprising: Administering to the subject a prophylactically and / or therapeutically effective amount of Aβo*3F according to claim 1 or 2, a composition according to any one of claims 3 to 6, 14 and 15, or a binding agent according to claim 10 or 11. A method for preventing and / or treating MCI and / or AD in a subject.

18. Use of Aβo*3F as a target for diagnosing MCI and / or AD in a subject, The Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody or its variant in a subject, and based on analysis by size exclusion chromatography (SEC), its total molecular weight is approximately 588 kDa. The light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively. The variant is a K98R variant or a 7B antibody, and its light chain CDR sequence and heavy chain CDR sequence are shown in SEQ ID NOs: 25-30 or SEQ ID NOs: 32-35 and KAS, respectively. Use of Aβo*3F.

19. 1. Use of Aβo*3F as a target for preventing and / or treating MCI and / or AD in a subject, The Aβo*3F is an Aβ oligomer that specifically binds to the 3F antibody or its variant in a subject, and based on analysis by size exclusion chromatography (SEC), its total molecular weight is approximately 588 kDa. The light chain CDR sequence and heavy chain CDR sequence of the 3F antibody are shown in SEQ ID NOs: 17-22, respectively. The variant is a K98R variant or a 7B antibody, and its light chain CDR sequence and heavy chain CDR sequence are shown in SEQ ID NOs: 25-30 or SEQ ID NOs: 32-35 and KAS, respectively. Use of Aβo*3F.

20. The subject is a human, a non-human primate, a cat, or a dog.

20. The use according to claim 15, the Aβo*3F, composition or binding agent according to claim 16, the method according to claim 17, or the use according to claim 18 or 19.

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