Improved amyloid beta oligomer-specific binding antibodies

Abstract

The present invention relates to an improved beta amyloid oligomer (A [beta] Os) specific binding antibody. Specifically, the invention relates to an improved form of a W20 antibody, the affinity of the improved form of the W20 antibody with AbetaOs is remarkably improved compared with that of the W20 antibody, Abeta aggregation and AbetaOs-induced nerve cell toxicity can be more remarkably inhibited, the cognitive and memory functions of an Alzheimer disease model mouse are more effectively improved, and pathological changes in the brain of the mouse are reduced. The antibody in an improved form can be specifically combined with oligomers of beta amyloid protein, alpha-synuclein, mHTT and SOD1, inhibits aggregation and cytotoxicity of each amyloid protein, has better potential for treating various amyloid protein diseases such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and the like compared with a W20 antibody, and can be used for preparing an antibody for treating various amyloid protein diseases such as Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. The improved antibody can be specifically combined with a strong-toxicity amyloid protein oligomer A beta o * 3F and has a better AD diagnosis value, and the amino acid sequence of the W20 antibody is as shown in SEQ ID No.1.

Description

Improved beta-amyloid oligomer-specific binding antibodies Technical Field

[0001] The present invention relates to antibodies that specifically bind to amyloid β oligomers (AβOs), and in particular to improved antibodies that specifically bind to specific AβOs. Background Art

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease that commonly affects the elderly. Its clinical symptoms primarily include memory loss, cognitive impairment, and loss of self-care abilities. AD is ranked as the fourth leading cause of death in humans, second only to cancer, heart disease, and stroke, and its incidence is significantly age-dependent. With the aging of the global population, the number of people suffering from AD is increasing annually. According to statistics, there were approximately 50 million AD patients worldwide in 2018, and the number is expected to reach 130 million by 2050. AD medical costs are enormous. In 2015, global spending on treatment and care for AD patients reached approximately US$818 billion, a figure projected to reach US$2 trillion by 2030. Over the past decade, approximately US$110 billion has been invested globally in drug research and development for AD treatments. However, despite this enormous investment, specific therapeutic agents have yet to be developed, and hundreds of therapeutic agents have failed in clinical trials. Developing drugs that target the pathogenesis of AD has become a hot research topic for many international pharmaceutical companies and research institutions.

[0003] Alzheimer's disease (AD) is a neurodegenerative disease in the elderly characterized by memory loss, the formation of senile plaques in the brain, and the formation of neural tangles within neurons. It is generally believed that the most cytotoxic Aβ oligomers, rather than Aβ monomers and fibrils, are the primary driver of AD. The level of Aβ oligomers in the brain of AD patients is highly correlated with the onset, progression, and severity of AD. Aβ oligomers have a high affinity for synapses, triggering the redistribution of key synaptic proteins and inducing overactivation of synaptic glutamate receptors. They can also damage neuronal membranes, disrupt synaptic Ca2+ homeostasis, and increase intracellular oxidative stress and mitochondrial damage. Binding of Aβ oligomers to synapses activates the complement system, prompting microglial cells to excessively prune and phagocytose synapses, disrupting synaptic structure and inducing synaptic dysfunction, leading to synaptic damage and excessive loss, and ultimately, cognitive decline. Therefore, targeting Aβ oligomers and inhibiting their levels and the neurotoxicity they induce is an ideal strategy for treating the cause of AD. However, treatments targeting Aβ monomers, particularly immunotherapies targeting Aβ monomers, can lead to adverse reactions such as inflammation and synaptic loss, leading to repeated failures in clinical trials. Clearing Aβ and reducing Aβ oligomer levels through immunotherapy strategies has been a research focus in the field of AD treatment. In recent years, dozens of antibodies and vaccines targeting Aβ have entered clinical trials. These include AN1792, a first-generation vaccine targeting Aβ fibrils, and antibodies targeting Aβ monomers or fibrils, such as bapineuzumab (anti-Aβ1-5), solanezumab (anti-Aβ13-28), gantenerumab (anti-Aβ1-11), and ponezumab (anti-Aβ C-terminus). Although 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 animals' brains, few have passed Phase III clinical trials in AD clinical trials due to poor therapeutic effects or serious side effects. In June 2021, the US FDA approved the Aduhelm (aducanumab) antibody for marketing under an accelerated approval program. Aducanumab can specifically recognize Aβ oligomers and has shown certain efficacy in clinical trials, but the antibody still has adverse reactions such as brain edema and inflammation, and its efficacy is also controversial.

[0004] Many neurodegenerative diseases are characterized by the abnormal aggregation of amyloid proteins, such as Aβ, tau, α-synuclein, mHTT, and SOD1. Oligomers formed by the aggregation of these protein monomers are key pathogenic factors in the development and progression of diseases such as AD, Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), and are effective targets for the diagnosis and treatment of these diseases. Oligomers and fibrils of different amyloid proteins are deposited in different parts of the brain, damaging neurons in corresponding areas and leading to cognitive and behavioral impairments. Although these amyloid proteins have different primary sequences, their aggregated oligomers can form similar spatial structures and share similar toxic mechanisms. Therefore, therapeutic strategies targeting toxic amyloid oligomers are of great significance for the diagnosis and treatment of various neurodegenerative diseases. Furthermore, studies have demonstrated that various amyloid proteins interact synergistically in amyloid diseases. The aggregation and progression of one amyloid protein can promote or induce the aggregation of other amyloid proteins and contribute to the pathological process of the disease. For example, clinical studies have found that in addition to the deposition of Aβ plaques and neurofibrillary entanglements caused by tau protein in the brains of 50% of AD patients, there are also obvious Lewy bodies (LB) produced by the aggregation and deposition of α-synuclein. Compared with AD patients without LB deposition, these patients have more severe pathological features and decreased cognitive function. Transgenic animal experiments have shown that Aβ, tau and α-synuclein can synergize with each other, promote each other's aggregation and deposition, and accelerate pathological changes and cognitive impairment in the brains of transgenic mice. Therefore, treatments targeting only one amyloid protein may not be enough to cure amyloid disease. Preparations that specifically target the structural features shared by multiple amyloid oligomers without relying on their primary amino acid sequence will become an ideal strategy for treating one or more amyloid diseases.

[0005] Since 2005, the inventors have conducted research on amyloid oligomer-specific antibodies and were the first in the world to screen and obtain a fully human single-chain antibody W20 that can recognize multiple amyloid oligomers (Chinese patent CN101463082A). This antibody can bind to oligomers such as Aβ, α-synuclein, mHTT, and SOD1, which cause various amyloid protein diseases such as AD, PD, HD and ALS, but not to monomers and fibers of each amyloid protein; this antibody can also inhibit the aggregation and cytotoxicity of various amyloid proteins; improve the cognitive function and behavioral coordination ability of disease model mice, and reduce neuropathological changes in mice (Biochimica et Biophysica Acta 2011, 1814, 1703-1712; AD: Current Alzheimer Research, 2014, 11, 69-78; PD&HD: Scientific Reports, 2016, 6, 36631; ALS: International Immunopharmacology 2018, 65, 413-421).

[0006] To date, there is still an urgent need in this field to further enhance the drugability of amyloid oligomer-specific antibodies and to develop antibody drugs that can effectively treat and / or prevent neurodegenerative diseases.

[0007] Summary of the Invention

[0008] One aspect of the present invention relates to an improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, which has an amino acid substitution at one or more positions selected from amino acid residue positions 226, 227 and 228 relative to the W20 antibody, the amino acid sequence of the W20 antibody being shown in SEQ ID No. 1, and the amino acid position numbering of the antibody being determined using the Kabat numbering system.

[0009] In some embodiments, the amino acid residue at position 226 of the improved amyloid oligomer-specific antibody or its antigen-binding fragment is substituted with a non-polar, hydrophobic amino acid with properties similar to alanine, preferably, substituted with glycine, valine, leucine, isoleucine, phenylalanine, tryptophan or proline, more preferably, substituted with glycine, phenylalanine or tryptophan.

[0010] In some embodiments, the amino acid residue 227 of the improved amyloid oligomer-specific antibody or its antigen-binding fragment is substituted with a hydrophobic amino acid having properties similar to valine, preferably, substituted with phenylalanine, tryptophan, tyrosine, alanine, leucine or isoleucine, more preferably, substituted with phenylalanine, isoleucine or leucine.

[0011] In some embodiments, the amino acid residue 228 of the improved amyloid oligomer-specific antibody or its antigen-binding fragment is substituted with an aliphatic amino acid having a structure similar to arginine, preferably, substituted with alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, glycine, serine, threonine, cysteine, asparagine or glutamine, more preferably, substituted with lysine, asparagine or glutamine.

[0012] In some embodiments, amino acid residues 226-228 of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof are substituted with AVR, GSR, WVR, FER, NFR or VRR, respectively.

[0013] In some embodiments, the amino acid residues 224 and 225 of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof are substituted with glutamine and threonine, respectively.

[0014] In some embodiments, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof can specifically bind to Aβo*3F oligomers.

[0015] In some embodiments, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is an antigen-binding fragment, preferably, an antigen-binding fragment selected from the group consisting of scFv, F(ab')2, Fab', Fab, Fd, Fv, bispecific antibody, camel antibody, CDR and antibody minimal recognition unit (dAb), more preferably, an antigen-binding fragment selected from the group consisting of scFv, F(ab')2, Fab', Fab. In some embodiments, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is scFv or F(ab')2.

[0016] In some embodiments, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. Preferably, the antibody is selected from the group consisting of IgG, IgM, IgA, IgD, and IgE, and their subtypes. More preferably, the antibody is selected from the group consisting of subtypes IgG1-4, and more preferably, the antibody is an IgG4 subtype. In some embodiments, the heavy chain amino acid sequence of the antibody is as shown in any one of SEQ ID No. 18-21, and / or the light chain amino acid sequence of the antibody is as shown in SEQ ID No. 17. In some embodiments, the heavy chain constant region amino acid sequence of the antibody is as shown in any one of SEQ ID No. 23-26, and / or the light chain constant region amino acid sequence of the antibody is as shown in SEQ ID No. 22.

[0017] In some embodiments, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is scFv and its amino acid sequence is shown in any one of SEQ ID No. 5, 4 and 6-9.

[0018] In some embodiments, the sequence of CDR3 of the light chain of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is shown as SEQ ID No.15.

[0019] In some embodiments, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is substituted with arginine at position 98 relative to the 3F antibody. In some embodiments, the heavy chain amino acid sequence of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID Nos. 33-36, and the light chain amino acid sequence is as described above. In some embodiments, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is a scFv and its amino acid sequence is as shown in SEQ ID No. 27.

[0020] In some embodiments, the present invention relates to a variant of the improved amyloid oligomer-specific antibody or its antigen-binding fragment as described above, which has a substitution, insertion, or deletion of one or more amino acid residues relative to the improved amyloid oligomer-specific antibody or its antigen-binding fragment as described above, such as a substitution, insertion, or deletion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 or more amino acid residues, while still substantially retaining one, multiple or all biological activities of the improved amyloid oligomer-specific antibody or its antigen-binding fragment as described above. In some embodiments, the substitution of the amino acid residue is a substitution of a conservative amino acid residue. In some embodiments, the present invention relates to a variant of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof as described above, which has at least 85% identity with the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof as described above, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.3%, 99.6% or higher identity, while still substantially retaining one, more or all biological activities of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof as described above. It is known to those skilled in the art that the above-mentioned identity is calculated based on a single amino acid residue, and therefore, the above-mentioned numerical values ​​are only approximate values. Those skilled in the art can accurately calculate the corresponding identity percentage based on the number of actual amino acid residue substitutions, insertions or deletions.

[0021] Another aspect of the present invention relates to an isolated nucleic acid molecule selected from the group consisting of:

[0022] (1) DNA or RNA encoding the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof as described above;

[0023] (2) A nucleic acid molecule that is completely complementary to the DNA or RNA defined in (1).

[0024] A further aspect of the present invention relates to an expression vector comprising an operably linked nucleic acid molecule as described above.

[0025] Yet another aspect of the present invention relates to a host cell comprising the nucleic acid molecule or expression vector described above.

[0026] Another aspect of the present invention relates to a composition comprising the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector or host cell as described above, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0027] Yet another aspect of the present invention relates to a method for producing the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof as described above, comprising the steps of:

[0028] The host cells described above are cultured under culture conditions suitable for the expression of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, and optionally, the resulting product is isolated and purified.

[0029] Yet another aspect of the present invention relates to a composition comprising the improved amyloid oligomer-specific antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell as described above, or the improved amyloid oligomer-specific antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell obtained by the method as described above, and another neurodegenerative disease therapeutic agent. Preferably, the other neurodegenerative disease therapeutic agent is selected from acetylcholinesterase inhibitors such as donepezil, galantamine, carbastine, etc., and aspartate receptor antagonists such as memantine, etc.

[0030] Another aspect of the present invention relates to the use of the improved amyloid oligomer-specific antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell or composition in the preparation of a medicament for inhibiting the aggregation of Aβ and / or Aβ oligomer-induced cytotoxicity in a subject, or for treating and / or preventing a neurodegenerative disease in a subject, or for diagnosing whether a subject has a neurodegenerative disease.

[0031] In some embodiments, the improved amyloid oligomer-specific antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell or composition is used to inhibit the aggregation of Aβ and / or Aβ oligomer-induced cytotoxicity in a subject, or to treat and / or prevent a neurodegenerative disease in a subject, or to diagnose whether a subject has a neurodegenerative disease.

[0032] Another aspect of the present invention relates to a method for inhibiting the aggregation of Aβ and / or the cytotoxicity induced by Aβ oligomers in a subject, or treating and / or preventing a neurodegenerative disease in a subject, or diagnosing whether a subject has a neurodegenerative disease, which comprises administering a therapeutically effective amount of the improved amyloid oligomer-specific antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell or composition as described above to the subject or the subject's cells.

[0033] In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia or spinocerebellar ataxia, preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis.

[0034] Another aspect of the present invention relates to the use of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition in the preparation of a reagent for diagnosing the presence and / or level of toxic forms of amyloid in a subject sample.

[0035] Another aspect of the present invention relates to the use of the improved amyloid oligomer-specific antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, host cell or composition as described above in the preparation of a drug for specifically binding to the highly toxic amyloid oligomer Aβo*3F in a subject and inhibiting its neurotoxicity, wherein the highly toxic amyloid oligomer Aβo*3F is separated from the Aβ oligomer mixture by immunoprecipitation against 3F antibody, and its typical characteristics are Aβ high molecular weight oligomers, and based on molecular exclusion chromatography (SEC) analysis, its molecular weight is about 588 kDa and its diameter is about 10 nm.

[0036] In some embodiments, the amyloid protein is selected from the group consisting of beta-amyloid, alpha-synuclein, mHTT, and SOD1.

[0037] In other words, to enhance the drugability of the antibody, the present invention subjected the W20 antibody to mature mutational engineering, with the primary mutation site located in the light chain CDR3 region, for example, by mutating the QTHRP sequence to the NSAVR sequence. Molecular modeling experiments demonstrated that the improved antibodies of the present invention, such as Antibody 3F, can recognize specific structures within oligomers formed by aggregated amyloid proteins. Furthermore, the improved antibodies, such as Antibody 3F, exhibit significantly enhanced binding affinity for Aβ oligomers and significantly prolonged their in vivo half-life. 3F inhibits the aggregation and cytotoxicity of multiple amyloid proteins, including Aβ, α-synuclein, and mHTT, in vitro. Intranasal administration for 20 days at a microgram-level dose significantly improved memory in AD transgenic mice and reduced brain pathological changes and inflammatory responses. Acute toxicity studies using an antibody dose 1000 times greater than the therapeutic dose demonstrated good animal tolerance, with no pathological changes observed in organs or tissues. Therefore, 3F demonstrates significant therapeutic efficacy, good safety, and excellent stability, demonstrating promising drugability.

[0038] More significantly, the Aβ oligomers specifically recognized by this antibody are highly toxic oligomers, the most predominant toxic component of the Aβ oligomer mixture, with potent pathogenicity and a key role in the development and progression of AD. The highly toxic oligomers recognized by 3F are present in the CSF, blood, and / or brain tissue of AD patients and AD-derived MCI patients, and their levels in the CSF, blood, and / or brain of these three groups of people show significant differences, allowing precise differentiation between AD patients, MCI patients, and healthy elderly populations. These toxic oligomers are also present in AD transgenic mice and are directly associated with the onset of AD in these mice. In the present invention, the highly toxic Aβ oligomer recognized by 3F is referred to as AβO*3F. It can be isolated from a mixture of Aβ oligomers using immunoprecipitation (3F antibody). Its typical characteristics are high-molecular-weight Aβ oligomers. Based on molecular exclusion chromatography (SEC) analysis, its molecular weight is approximately 588kDa and its diameter is approximately 10nm. It has a strong toxic effect on neurons and is more than 200 times more toxic than the Aβ oligomer mixture. Aβo*3F can activate microglia and / or astrocytes, secreting large amounts of inflammatory factors. More importantly, the present invention prepared Aβo*3F in vitro. The Aβo*3F prepared in vitro is consistent in composition with the product immunoprecipitated from the cerebrospinal fluid or plasma of AD patients using the 3F antibody, and has the same physicochemical properties and functions. Although some prior art mAbs such as lecanemab also target Aβ aggregates, the Aβ aggregates they target are different from the Aβ aggregates targeted by the improved antibodies of the present invention such as antibody 3F, which specifically targets the highly neurotoxic oligomer Aβo*3F as described above.

[0039] Specifically, the improved antibodies of the present invention, such as antibody 3F, have the following advantages and characteristics:

[0040] 1) Compared with the W20 antibody, the affinity between 3F and the highly toxic amyloid protein oligomer Aβo*3F is significantly improved, which can better inhibit the aggregation and cytotoxicity of amyloid protein and has better therapeutic potential.

[0041] 2) 3F specifically binds to the highly toxic amyloid oligomer Aβo*3F, which is present in the cerebrospinal fluid (CSF), blood, and / or brain tissue of AD patients and patients with AD-induced mild cognitive impairment (MCI). Levels in the CSF, blood, and / or brain of AD patients, MCI patients, and healthy elderly individuals show extremely significant differences. Aβo*3F is an extremely toxic oligomer and the most predominant toxic component in the Aβ oligomer mixture. It has a strong pathogenic effect and plays a key role in the occurrence and development of AD. Therefore, Aβo*3F can be used as a target for the diagnosis, prevention, and / or treatment of MCI and / or AD in subjects. 3F targeting Aβo*3F has good application value in the diagnosis and treatment of AD.

[0042] 3) 3F specifically binds to the highly toxic amyloid oligomer Aβo*3F, a key pathogenic factor, does not recognize monomers and fibers, and does not cause autoimmune reactions. Because amyloid monomers, such as Aβ and α-synuclein, have normal physiological functions in the body, antibodies that bind to these monomers are prone to cause autoimmune reactions and reduce the effective concentration of the antibody target in the body. This is also one of the main reasons why many antibodies and vaccine drugs have failed in clinical trials. In addition, most of the dozens of antibodies and vaccines targeting Aβ that have entered the clinical trial stage have poor therapeutic effects or serious adverse reactions. Studies have shown that because many antibodies target Aβ monomers and their antigen-antibody complexes, they will trigger an inflammatory response and cause synaptic loss, resulting in the failure of Aβ-targeted immunotherapy for AD. The 3F antibody can overcome this side effect and achieve a better AD treatment effect.

[0043] 4) 3F can bind to a variety of amyloid proteins with different primary sequences, reducing their toxicity and promoting their clearance. Amyloid proteins with different primary structures can form similar structures when aggregated, such as being rich in β-sheet structures and able to bind to thioflavin (ThT) and Congo red. This forms the basis for 3F's binding to a variety of amyloid oligomers. 3F can specifically bind to oligomers of Aβ, α-synuclein, mHTT, and SOD1, and inhibit the aggregation and cytotoxicity of each amyloid protein. Compared with W20, 3F has a more significant therapeutic effect on model animals such as AD, PD, HD, and ALS, and has greater application and translational potential.

[0044] 5) The therapeutic dosage of 3F is small, the therapeutic effect is obvious, the therapeutic mechanism is clear, the safety is high, and the properties are stable.

[0045] 6) Since Aβo*3F is an oligomer formed by the aggregation of Aβ monomers, all antibodies targeting Aβ monomers may bind to Aβo*3F. However, compared with the antibodies of the present invention, such as the 3F antibody, which specifically recognize and bind to Aβo*3F, antibodies targeting Aβ monomers in the prior art can also bind to various forms such as Aβ monomers, oligomers, and fibers. Therefore, their chance of binding to the highly neurotoxic oligomer Aβo*3F of the present invention is greatly reduced, and accordingly, their efficacy is also dispersed, resulting in a decrease in the AD treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1: Shows the phage antibody library polyclonal ELISA.

[0047] Figure 2: shows phage monoclonal ELISA.

[0048] Figure 3 shows the structural molecular simulation of the single-chain antibody W20 and its key amino acid residues.

[0049] Figure 4 shows that the 3F antibody specifically binds to Aβ oligomers. Dot blot analysis was used to examine the binding ability of the 3F antibody to Aβ42 monomers and aggregates.

[0050] Figure 5: shows that 3F antibody can significantly inhibit Aβ aggregation and Aβ oligomer-induced cytotoxicity:

[0051] A. Thioflavin T (ThT) assay to detect the inhibitory effects of 3F and W20 antibodies on Aβ aggregation;

[0052] B. MTT assay was used to detect the inhibitory effects of 3F and W20 antibodies on Aβ oligomer-induced neuronal cytotoxicity.

[0053] Figure 6: Shows that 3F antibody can significantly improve the cognitive function of AD transgenic mice:

[0054] A. The latency of mice in each group to find the platform during the training period of the water maze experiment;

[0055] B. The number of times the mouse crossed the platform during the exploration period after the platform was removed in the water maze experiment;

[0056] The time that mice in each group stayed in the new arm in the CY maze experiment.

[0057] Figure 7: shows that 3F antibody can significantly reduce the level of Aβ plaques in the brains of AD transgenic mice:

[0058] A. Immunostained APP / PS1 mouse brain slices using 4G8 antibody to detect senile plaque levels in the mouse brain;

[0059] B. Quantitative statistics of the senile plaque area in the cortex and hippocampus of mice.

[0060] Figure 8 shows that 3F antibody can significantly reduce Aβ levels in the brains of AD transgenic mice. ELISA was used to measure the levels of Aβ40 / 42 in brain homogenates of APP / PS1 mice.

[0061] Figure 9: 3F antibody can significantly reduce the activation of glial cells in the brains of AD transgenic mice.

[0062] A. Iba-1 and GFAP immunostaining were used to detect the activation of microglia and astrocytes in the brains of APP / PS1 mice.

[0063] B. Quantitative statistics of Iba-1 and GFAP positive staining areas in the cortex and hippocampus of mouse brain.

[0064] Figure 10 shows that 3F antibody can significantly reduce the levels of inflammatory factors in the brain of AD transgenic mice. ELISA was used to measure the levels of IL-1β and IL-6 in brain homogenates of APP / PS1 mice.

[0065] Figure 11 shows that 3F antibody can significantly improve the behavioral coordination and cognitive function of PD transgenic mice:

[0066] A. The time taken by mice in each group to turn their heads in the pole climbing test;

[0067] B. The time taken by mice in each group to climb into the cage in the pole climbing test;

[0068] C. Cognitive index of each group of mice in the novel object recognition experiment.

[0069] Figure 12 shows that 3F antibody can significantly reduce the level of α-synuclein in the brain of PD transgenic mice:

[0070] A. Immunostained brain slices of A53T α-synuclein transgenic mice using pSer129-α-syn antibody to detect pathological α-synuclein levels in the mouse brain;

[0071] B. Quantitative statistics of the pSer129-α-syn positive staining area in the mouse brainstem region.

[0072] Figure 13: 3F antibody can significantly increase the level of tyrosine hydroxylase in the brain of PD transgenic mice:

[0073] A. Immunostained brain slices of A53T α-synuclein transgenic mice using tyrosine hydroxylase (TH) antibody to detect TH levels in the mouse brain;

[0074] B. Quantitative statistics of TH-positive staining area in the mouse brainstem.

[0075] Figure 14 shows that 3F antibody can significantly improve the spontaneous activity and anxiety behavior of HD transgenic mice: statistical analysis of the total distance traveled (A), the number of standing times in all areas (B), and the time spent in the central area (C) of each group of mice in the open field test.

[0076] Figure 15: shows that 3F antibody can significantly reduce the level of mHTT aggregates in the brain of HD transgenic mice:

[0077] A. Immunostained R6 / 2 mouse brain slices using EM48 antibody to detect the level of mHTT aggregates in the mouse brain;

[0078] B. Quantitative statistics of EM48-positive staining area in mouse brain.

[0079] Figure 16 shows that 3F antibody can significantly improve the motor function of ALS transgenic mice:

[0080] A. Stay time of mice in each group in the hanging experiment;

[0081] B. The duration of each group of mice on the rotarod in the rotarod test.

[0082] Figure 17 shows that 3F antibody can significantly reduce the level of SOD1 aggregates in the brainstem of ALS transgenic mice:

[0083] A. Immunostained SOD1-G93A mouse brain slices using SOD1 antibody to detect the level of SOD1 aggregates in the mouse brain;

[0084] B. Quantitative statistics of SOD1-positive staining area in mouse brainstem.

[0085] Figure 18 shows that 3F antibody can significantly reduce the activation of glial cells in the brains of ALS transgenic mice:

[0086] A. Iba-1 and GFAP immunostaining were used to detect the activation of microglia and astrocytes in the brains of SOD1-G93A mice.

[0087] B. Quantitative statistics of Iba-1 and GFAP positive staining areas in mouse brain.

[0088] Figure 19 shows that antibodies can improve cognitive function in AD transgenic mice. The time that each group of APP / PS1 mice spent in the novel arm in the Y-maze test was compared.

[0089] FIG20 shows the results of sequence alignment of positive clones with that of W20.

[0090] FIG. 21 shows that the 3FI4 antibody (IgG4 subtype of the A16 antibody) can specifically bind to Aβ oligomers.

[0091] FIG22 shows a schematic diagram of immunoprecipitation preparation of Aβo*3F, Aβ*6E10 and Aβ-ID.

[0092] Figure 23 shows the molecular weight, morphology and cytotoxicity of Aβo*3F.

[0093] A: Size exclusion chromatography (SEC) analysis of mAβo*3F molecular weight; SEC analysis (Superdex 200 10 / 300) of mAβo*3F and SEC standards. SEC standards: 1. thyroglobulin (669 kDa), 2. ferritin (440 kDa), 3. aldolase (158 kDa), 4. conalbumin (75 kDa), 5. ovalbumin (44 kDa), 6. carbonic anhydrase (29 kDa).

[0094] B: IC50 of sAβos cytotoxicity to N2a cells;

[0095] C: IC50 of sAβo*3F cytotoxicity to N2a cells;

[0096] D: IC50 of mAβo*3F toxicity to N2a cells and primary neurons;

[0097] E: IC50 of hAβo*3F toxicity to primary neurons.

[0098] Figure 24 shows that Aβo*3F significantly reduces cognitive function and damages neurons in the mouse brain:

[0099] A: The duration of each group of mice in the novel arm in the Y-maze test;

[0100] B: Golgi staining to detect the density of dendritic spines in mouse neurons;

[0101] C: Statistical analysis of dendritic spine density in B.

[0102] Figure 25 shows that after the 98th position K of the single-chain antibody heavy chain was mutated to R, the affinity was further improved, suggesting that the 98th amino acid plays an important role in the binding of the single-chain antibody to Aβ oligomers. DETAILED DESCRIPTION

[0103] definition

[0104] Amyloid proteins include β-amyloid protein, microtubule-associated protein tau, α-synuclein, huntingtin protein, pancreatic amyloid protein, superoxide dismutase 1 (SOD1) and TDP-43 protein, including their aggregated forms such as monomers, oligomers, pre-fibrils or fibrils.

[0105] Toxic forms of amyloid protein refer to forms of amyloid protein that play a negative role in the occurrence and development of neurodegenerative diseases, such as oligomeric forms and profibrillar forms.

[0106] Amyloid oligomers refer to non-fibrillar aggregates formed by the aggregation of two or more amyloid monomer molecules.

[0107] The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule that has the ability to bind to an epitope on an antigen. Naturally occurring antibodies typically comprise tetramers, which are generally composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the following isotypes: IgG, IgA, IgM, IgD, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Igs of the same class are further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of disulfide bonds in their heavy chains. For example, IgG is divided into IgG1, IgG2, IgG3, and IgG4 subclasses, and IgA is divided into IgA1 and IgA2 subclasses. Light chains are divided into kappa and lambda chains based on differences in their constant regions. The antibodies of the present invention may have any isotype. The choice of isotype is generally determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant domains κ or λ can be used. If necessary, the class of the antibody of the present invention can be converted by known methods. For example, an antibody of the present invention that was originally an IgG can be converted to an IgM antibody of the present invention by class conversion. In addition, class conversion technology can be used to convert one IgG subclass into another subclass, for example, from IgG1 to IgG2. Therefore, the effector function of the antibody of the present invention can be converted to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies by isotype switching for various therapeutic uses, provided that the complement of the antibody, such as C1q and / or Fc receptor binding activity, is reduced or eliminated. In some embodiments, the antibody of the present invention is an IgM or IgG1, 2, 3 or 4 type antibody. If the amino acid sequence of the antibody is largely homologous to the isotype relative to other isotypes, the antibody belongs to a specific isotype.

[0108] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to complete antibodies and antigen-binding fragments of antibodies.

[0109] A "variable region" or "variable domain" is a domain in an antibody's heavy or light chain that participates in binding the antibody to its antigen. Each heavy chain of an antibody is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which is typically composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The heavy and light chain variable regions are typically responsible for antigen recognition, while the heavy and light chain constant domains can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), Fc receptors, and the first component (C1q) of the classical complement system. The heavy and light chain variable regions contain a binding region that interacts with an antigen. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) called "complementarity determining regions (CDRs)," which are interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDR domains and four FR domains, arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0110] The term "complementarity determining region" or "CDR region" or "CDR" (used interchangeably herein with hypervariable region "HVR") refers to the region in the antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigenic epitopes. In this article, the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0111] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0112] The term "monoclonal antibody", "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule preparation of a single molecular composition, which refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the group comprising individual antibodies is identical except for possible naturally occurring mutations that may be present in small amounts. Conventional monoclonal antibody compositions exhibit a single binding specificity and affinity for a specific epitope. In certain embodiments, a monoclonal antibody can be composed of more than one Fab domain, thereby increasing specificity for more than one target. The term "monoclonal antibody" or "monoclonal antibody composition" is not limited to any specific production method (e.g., recombinant, transgenic, hybridoma, etc.).

[0113] The present invention also includes "bispecific antibodies," wherein the antibodies of the present invention are part of a bivalent or multivalent bispecific framework that targets more than one epitope (e.g., the second epitope can comprise an epitope of an active transport receptor, such that the bispecific antibody exhibits improved transcytosis across biological barriers (e.g., the blood-brain barrier)). Thus, in another embodiment, a monovalent Fab of an antibody of the present invention can be linked to another Fab or scFv targeting a different protein to produce a bispecific antibody. Bispecific antibodies can have dual functions, such as a therapeutic function conferred by the present invention and a transport function that can bind to a receptor molecule to enhance translocation across a biological barrier (e.g., the blood-brain barrier). The terms "bispecific antibody," "bifunctional antibody," "bispecific antibody," "bispecific antibody," or "BsAb" refer to antibodies that possess two different antigen-binding sites, thereby being able to bind to two target antigens simultaneously, mediating another specific function while exerting the antibody's targeting properties. The specific function effector molecule mediated can also be a toxin, enzyme, cytokine, radionuclide, etc. The two arms of the bispecific antibody that bind to the antigen can be derived from Fab, Fv, ScFv, or dSFv, etc.

[0114] The term "antigen-binding fragment of an antibody" refers to a fragment, part, region or domain of an antibody that can bind to an epitope (e.g., obtainable via cleavage, recombination, synthesis, etc.). An antigen-binding fragment may contain 1, 2, 3, 4, 5 or all 6 CDR domains of such an antibody, and despite being able to bind to the epitope, may exhibit different specificities, affinities or selectivities. Preferably, the antigen-binding fragment contains all 6 CDR domains of the antibody. An antigen-binding fragment of an antibody may be a part of or comprise a single polypeptide chain (e.g., scFv), or may be a part of or comprise two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., diabodies, Fab fragments, F(ab')2 fragments, etc.).

[0115] Examples of antigen-binding fragments encompassed by the present invention include (a) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (b) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge domain; (c) Fd fragments consisting of the VH region and the CH1 domain; (d) Fv fragments consisting of the VL and VH regions of a single antibody arm; (e) single-chain antibodies (scFv), recombinant proteins formed by genetically engineering the VH and VL regions of an antibody linked by a linker peptide; (f) dAb fragments (Ward et al., Nature, 341, 544-546 (1989)), which consist essentially of the VH region and are also known as domain antibodies (Holt et al., Trends Biotechnol., 2i(11):484-90); (g) camelid or nanobodies (Revets et al., Expert Opin Biol., 21(11):509-610); Ther., 5(l): 111-24) and (h) isolated complementarity determining regions (CDRs).

[0116] In some embodiments, the antibodies of the present invention and their antigen-binding fragments are single-chain antibodies. In some embodiments, the present invention provides single-chain Fv (scFv), wherein the heavy chain and light chain in the Fv of the antibody of the present invention are connected into a single peptide chain by a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues). The method for producing such antibodies is described in, for example, US 4,946,778; Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore ed., Springer-Verlag, New York, pp. 269-315 (1994); Bird et al., Science, 242, 423-426 (1988); Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature, 348, 552-554 (1990). Single-chain antibodies are monovalent if only a single VH and VL are used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used.

[0117] In some embodiments, the antibodies of the present invention and antigen-binding fragments thereof are chimeric antibodies. The term "chimeric antibody" refers to a portion of a heavy chain and / or light chain that is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass and a fragment of such an antibody, as long as they exhibit desired biological activity. The present invention provides variable region antigen-binding sequences from human antibodies. Therefore, the chimeric antibodies of primary concern herein include antibodies having one or more human antigen-binding sequences (such as CDRs) and containing one or more sequences such as FR or C region sequences from non-human antibodies. In addition, chimeric antibodies as described herein are antibodies comprising a human variable region antigen-binding sequence of an antibody class or subclass and another sequence such as FR or C region sequences from another antibody class or subclass.

[0118] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are humanized antibodies. The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

[0119] In some embodiments, the antibodies of the present invention and antigen-binding fragments thereof are human antibodies or fully human antibodies. The term "human antibody" or "fully human antibody" ("humAb" or "HuMab") refers to an antibody comprising variable and constant domains derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis or during gene rearrangement or in vivo by somatic mutation).

[0120] Variant antibodies are also included within the scope of the present invention. Thus, variants of the sequences listed in the application are also included within the scope of the present invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art and are also included within the scope of the present invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can be used to improve the translation efficiency of expression systems used in antibody production.

[0121] Such variant antibody sequences have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher) sequence homology with the sequences listed in the application. Such sequence homology is calculated relative to the full length of the reference sequence (i.e., the sequence listed in the application).

[0122] The numbering of the amino acid residues in the present invention is based on Or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C., (1991), Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia, C. & Lesk, AM, (1987), Canonical structures For The Hypervariable domains Of Immunoglobulins., J. Mol. Biol., 196, 901-917. Unless otherwise specified, the numbering of amino acid residues in the present invention is based on the Kabat numbering system.

[0123] When an antibody or antigen-binding fragment thereof "specifically" binds to a region of another molecule (i.e., an epitope), it means that it reacts or binds to that epitope more frequently, more rapidly, for a longer duration, and / or with greater affinity or avidity than to another epitope. In some embodiments, an antibody or antigen-binding fragment thereof of the invention binds to or binds to that epitope with at least 10 -7 The affinity of M for binding to human amyloid, especially its toxic forms, such as 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or higher. Preferably, the antibody or its antigen-binding fragment binds under physiological conditions (e.g., in vivo). Therefore, specific binding to amyloid, especially its toxic form, refers to the ability of the antibody or its antigen-binding fragment to bind to amyloid, especially its toxic form, with the above-mentioned specificity and / or under such conditions. Methods suitable for determining such binding are known in the art.

[0124] In the context of antibody binding to a specified antigen, the term "binding" generally refers to binding by an amount corresponding to about 10 -6 M or smaller K D The affinity binding, the K D The affinity of the antibody is at least 10-fold lower, such as at least 100-fold, at least 1,000-fold lower, than the affinity of the antibody for binding to a nonspecific antigen (e.g., BSA, casein) other than the designated antigen or a closely related antigen.

[0125] As used herein, the term "k d "(sec-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also called k off value.

[0126] As used herein, the term "k a "(M-1 x sec-1 or 1 / Msec) refers to the association rate constant for a specific antibody-antigen interaction.

[0127] As used herein, the term "K D "(M)" refers to the dissociation equilibrium constant for a specific antibody-antigen interaction and is expressed by k d Divide by k a get.

[0128] As used herein, the term "K A ” (M-1 or 1 / M) refers to the binding equilibrium constant for a specific antibody-antigen interaction and is expressed by k a Divide by k d get.

[0129] By taking advantage of the fact that a single amino acid change in a CDR residue can result in loss of functional binding (Rudikoff, S. et al., (1982), Single Amino Acid Substitution Altering Antigen-binding Specificity, Proc. Natl. Acad. Sci. (USA)) 79 (6): 1979-1983), alternative functional CDR sequences can be systematically identified. In a preferred method for obtaining such variant CDRs, a polynucleotide encoding a CDR is mutagenized (e.g., via random mutagenesis or by a site-directed approach) to generate a CDR having a substituted amino acid residue. By comparing the identity of the relevant residue in the original (functional) CDR sequence with the identity of the substituted (non-functional) variant CDR sequence, a BLOSUM62.iij substitution score for the substitution can be identified. The BLOSUM system provides amino acid substitution matrices created by analyzing sequence databases for reliable alignment (Eddy, SR, (2004), Where Did The BLOSUM62 Alignment Score Matrix Come From?, Nature Biotech., 22(8): 1035-1036; Henikoff, JG, (1992), Amino acid substitution matrices from protein blocks), Proc. Natl. Acad. Sci. (USA), 89: 10915-10919; Karlin, S. et al., (1990), Methods For Assessing The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes), PNAS, 87: 2264-2268; Altschul, SF, (1991), Amino Acid Substitution Matrices From An Information Theoretic Perspective, J. Mol. Biol., 219, 555-565. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 presents the BLOSUM62.iij substitution scores (the higher the score, the more conservative the substitution, and therefore the more likely it is that the substitution will not affect function).For example, if the antigen-binding fragment comprising the resulting CDR is unable to bind to PD-L1, the BLOSUM62.iij substitution score is considered insufficiently conservative, and new candidate substitutions with higher substitution scores are selected and generated. Thus, for example, if the original residue is glutamic acid (E) and the non-functional replacement residue is histidine (H), the BLOSUM62.iij substitution score will be 0, and more conservative changes (such as to aspartic acid, asparagine, glutamine, or lysine) are preferred.

[0130] The present invention therefore contemplates the use of random mutagenesis for identifying improved CDRs. In the context of the present invention, conservative substitutions can be defined by substitutions within the amino acid classes reflected in one or more of the following three tables:

[0131] Conservatively substituted amino acid residue categories:

[0132] Alternative conservative amino acid residue substitution categories:

[0133] Physical and functional substitutability of amino acid residues:

[0134] More conservative substitution groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0135] In some embodiments, the hydrophilic amino acid is selected from Arg, Asn, Asp, Gln, Glu, His, Tyr, and Lys.

[0136] Additional amino acid groupings may also be formulated using the principles described in, for example, Creighton, (1984), Proteins: Structure and Molecular Properties, WH Freeman and Company).

[0137] Thus, the sequence of the CDR variants of the included antibodies or antigen-binding fragments thereof may differ from the sequence of the CDRs of the parent antibody by substitutions; for example, substitutions of 4, 3, 2 or 1 amino acid residues. According to embodiments of the present invention, amino acids in the CDR regions may be substituted with conservative substitutions, as defined in the above three tables.

[0138] "Homology" or "sequence identity" refers to the percentage of residues in a polynucleotide or polypeptide sequence variant that are identical to the non-variant sequence after alignment and introduction of gaps. In specific embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.

[0139] Such variant polypeptide sequences have 70% or more (i.e. 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences listed in the application. In other embodiments, the invention provides polypeptide fragments comprising continuous stretches of various lengths of the amino acid sequences disclosed herein. For example, where applicable, the peptide sequences provided by the invention comprise peptides of at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more consecutive amino acids of one or more sequences disclosed herein, as well as peptides of all intermediate lengths therebetween.

[0140] The antibody of the present invention may be a monoclonal antibody produced by recombinant DNA.

[0141] In some embodiments, the antibodies of the present invention are full-length antibodies, preferably IgG1-4 antibodies or IgM antibodies. In other embodiments, the antibodies of the present invention are antibody antigen-binding fragments or single-chain antibodies.

[0142] The subunit structures and three-dimensional structures of the constant regions of various immunoglobulin classes are known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, while the term "CH1 domain" includes the first (most amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule. As used herein, the term "CH2 domain" includes a portion of a heavy chain molecule that ranges, for example, from about residue 244 to residue 360 ​​of an antibody using a conventional numbering scheme (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest." Interest" (1983). The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It has been documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues. The term "hinge region" as used herein includes that part of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen binding regions to move independently. The hinge region can be divided into three different domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161: 4083 (1998)).

[0143] In some embodiments, the antibodies of the present invention are monovalent antibodies, preferably monovalent antibodies with hinge region deletions as described in WO 2007059782 (incorporated herein by reference in its entirety). Thus, in some embodiments, the antibodies are monovalent antibodies, wherein the antibodies are constructed by the following method: i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VL region of the selected antigen-specific antibody and a nucleotide sequence encoding the constant CL region of Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of Ig are effectively linked together, and wherein, in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that in the presence of polyclonal human IgG or when administered to an animal or human, the CL region does not contain any amino acid that can form a disulfide bond or covalent bond with other peptides comprising the consistent amino acid sequence of the CL region; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the selected antigen-specific VL region and a nucleotide sequence encoding the constant CL region of Ig. The invention relates to a method for preparing a monovalent antibody comprising the steps of: comprising: preparing a nucleotide sequence of the VH region of a human antigen-specific antibody and a nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that, in the presence of polyclonal human IgG or when administered to an animal, the region corresponding to the hinge region and other regions of the CH region (such as the CH3 region) (as required by the Ig subtype) does not contain any amino acid residues that participate in forming disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising the consensus amino acid sequence of the CH region of a human Ig, wherein the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operatively linked together; iii) providing a cell expression system for producing a monovalent antibody; iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).

[0144] Similarly, in some embodiments, an antibody of the invention is a monovalent antibody comprising:

[0145] (i) the variable region of an antibody of the invention as described herein, or an antigen-binding portion of such a domain, and

[0146] (ii) a CH region or a domain thereof comprising a CH2 and CH3 domain of an immunoglobulin, wherein the CH region or domain thereof has been modified such that the domain corresponding to the hinge region and (if the immunoglobulin is not of the IgG4 subtype) the other domains of the CH region (such as the CH3 domain) does not contain any amino acid residues that are capable of forming disulfide bonds with the same CH region or forming other covalent or stable non-covalent inter-heavy chain bonds with the same CH region in the presence of polyclonal human IgG.

[0147] In other embodiments, the heavy chain of the monovalent antibody is modified such that the entire hinge region is deleted.

[0148] In further embodiments, the sequence of the monovalent antibody is modified such that it does not contain any acceptor sites for N-linked glycosylation.

[0149] The antibody of the present invention is produced by any technology known in the art, such as but not limited to any chemical, biological, genetic or enzymological technology, and can be used alone or in combination. Generally, knowing the amino acid sequence of the desired sequence, those skilled in the art can easily produce the antibody by the standard techniques for producing polypeptides. For example, these antibodies can be synthesized using known solid phase methods, preferably using commercially available peptide synthesis devices (such as the device manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions to synthesize these antibodies. Alternatively, the antibody of the present invention can be synthesized by recombinant DNA technology well known in the art. For example, after the DNA sequence encoding the antibody is incorporated into an expression vector and these vectors are imported into a suitable eukaryotic or prokaryotic host for expressing the desired antibody, the antibody as a DNA expression product can be obtained, and known techniques can be used afterwards to separate the antibody from the host.

[0150] The antibodies and antigen-binding fragments thereof of the present invention can be modified by comprising any "suitable" number of modified amino acids and / or in combination with coupling substituents. In this case, "suitable" is generally determined by the ability to at least substantially retain the selectivity of amyloid proteins, especially their toxic forms, and / or the specificity of amyloid proteins, especially their toxic forms, associated with the non-derivatized parent antibody. Including one or more modified amino acids can be advantageous in, for example, increasing the serum half-life of a polypeptide, reducing polypeptide antigenicity, or increasing polypeptide storage stability. One or more amino acids can be modified, for example, simultaneously with translation or after translation during recombinant production (e.g., N-linked glycosylation on NXS / T motifs during mammalian cell expression), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprenyl (e.g., farnesylation, geranyl-geranylation) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References to amino acid modifications are commonplace in the art, see, for example, Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. The modified amino acid can, for example, be selected from a glycosylated amino acid, a pegylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid coupled to a lipid moiety, or an amino acid coupled to an organic derivatizing agent.

[0151] The antibodies and antigen-binding fragments thereof of the present invention can also be chemically modified by covalent coupling to polymers to increase their circulation half-life. Exemplary polymers and methods of attaching them to peptides are described, for example, in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., having a molecular weight of between about 1,000 and 40,000 D, such as between about 2,000 and 20,000 D, such as about 3,000 to 12,000 D PEG).

[0152] The antibodies and antigen-binding fragments thereof of the present invention also include mutants formed by substitution, deletion or addition of one or more amino acids of the above-mentioned amyloid protein antibodies.

[0153] In another aspect, the present invention relates to expression vectors encoding one or more polypeptide chains of the antibodies or antigen-binding fragments thereof of the present invention. Such expression vectors can be used to recombinantly produce the antibodies and antigen-binding fragments thereof of the present invention.

[0154] In the present invention, the expression vector may be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (containing a nucleic acid sequence of a set of suitable expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, the nucleic acid encoding the antibody of the invention is contained in a naked DNA or RNA vector, including, for example, a linear expression element (as described in, for example, Sykes and Johnston, Nat Biotech, 12, 355-59 (1997)), a compact nucleic acid vector (as described in, for example, US 6,077,835 and / or WO 00 / 70087), a plasmid vector (such as pBR322, pUC 19 / 18 or pUC 118 / 119), a minimal size nucleic acid vector (as described in, for example, Schakowski et al., Mol Ther, 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4 precipitated construct (as described in, for example, WO 00 / 46147; Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986); Wigler et al., Cell, 14, 725 (1978) and Coraro and Pearson, Somatic Cell Genetics, 2, 603 (1981). Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0155] In some embodiments, the vector is suitable for expressing the antibody of the present invention or its antigen-binding fragment in bacterial cells. Examples of such vectors include, for example, BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem, 264, 5503-5509 (1989)), pET vectors (Novagen, Madison, Wisconsin), etc.

[0156] The expression vector may also be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience, New York (1987); Grant et al., Methods in Enzymol, 153, 516-544 (1987); Mattanovich, D. et al., Methods Mol. Biol., 824, 329-358 (2012); Celik, E. et al., Biotechnol. Adv., 30(5), 1108-1118 (2012); Li, P. et al., Appl. Biochem. Biotechnol., 142(2), 105-124 (2007); E. et al., Appl. Microbiol. Biotechnol., 77(3), 513-523 (2007); van der Vaart, JM, Methods Mol. Biol., 178, 359-366 (2002) and Holliger, P., Methods Mol. Biol., 178, 349-357 (2002)).

[0157] In the expression vector of the present invention, the nucleic acid encoding the antibody of the present invention can include any suitable promoter, enhancer and other elements that contribute to expression or be combined with it. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), effective poly (A) terminator sequences, origins of replication for producing plasmids in Escherichia coli, antibiotic resistance genes as selective markers and / or convenient cloning sites (e.g., polylinkers). Nucleic acid can also include an inducible promoter relative to a constitutive promoter (e.g., CMV IE).

[0158] In other aspects, the present invention relates to recombinant eukaryotic or prokaryotic host cells (such as transfectomas) that produce an antibody or antigen-binding fragment thereof of the present invention or a bispecific molecule of the present invention. Examples of host cells include yeast, bacteria, and mammalian cells (such as CHO or HEK cells). For example, in some embodiments, the present invention provides cells comprising a nucleic acid stably integrated into the cell genome, the genome comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof of the present invention. In other embodiments, the present invention provides cells comprising a non-integrated nucleic acid (such as a plasmid, cosmid, phagemid, or linear expression element) comprising a sequence encoding an antibody or antigen-binding fragment thereof of the present invention.

[0159] The antibodies and antigen-binding fragments thereof of the present invention can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines and insect cell lines, for example, CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (such as myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep. 18, 1-13., the contents of which are incorporated herein by reference).

[0160] The antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0161] The present invention further relates to compositions comprising, consisting of, or consisting essentially of an antibody of the present invention.

[0162] As used herein, "consisting essentially of" with respect to a composition means that at least one antibody of the present invention as described above is the only biologically active therapeutic agent or agent in the composition.

[0163] In one embodiment, the composition of the present invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient, diluent or carrier.

[0164] The present invention further relates to a medicament comprising, consisting of or consisting essentially of an antibody of the present invention and further comprising a pharmaceutically acceptable excipient, diluent or carrier.

[0165] The term "pharmaceutically acceptable carrier" refers to an excipient that does not produce adverse, allergic or other untoward reactions when administered to animals, preferably humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. For human administration, the formulation should meet the sterility, pyrogenicity, general safety and purity standards required by regulatory agencies (e.g., FDA Office or EMA).

[0166] In some embodiments, the glycosylation of the antibodies of the present invention is modified. For example, aglycosylated antibodies can be prepared (i.e., antibodies lacking glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen or alter the ADCC activity of the antibody. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is further described in detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al. (incorporated herein by reference). Additionally or alternatively, antibodies with altered glycosylation types can be prepared. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which the recombinant antibodies of the present invention are expressed, thereby producing antibodies with altered glycosylation.

[0167] The recombinant expression vector can be introduced into a host cell to produce a transformed host cell. The terms "transformed with," "transfected with," "conversion," "transduction," and "transfection" are intended to include introducing nucleic acids (e.g., vectors) into cells by one of many possible techniques known in the art. As used herein, the terms "transformed host cells" or "transduced host cells" are also intended to include cells transformed with the recombinant expression vectors of the present invention. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride-mediated transformation. For example, nucleic acids can be introduced into mammalian cells by conventional techniques, such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, liposome transfection, electroporation, or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, and other laboratory textbooks.

[0168] Although the nucleotide sequence defined above is DNA, in an alternative embodiment of the present invention, the nucleotide sequence may be RNA. Thus, corresponding RNA sequences to the DNA sequences described herein are contemplated. A skilled artisan will understand how to derive an RNA sequence encoding the same protein / polypeptide product as the DNA sequence shown above. "T" should be replaced with "U."

[0169] As used herein, the term "nucleic acid sequence" or "nucleic acid molecule" or "polynucleotide", "polynucleotide sequence" or "nucleotide sequence" refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes sequences containing modifications or substitutions of non-naturally occurring monomers or portions thereof. The nucleic acids, polynucleotides or nucleotide sequences of the present invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and can include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequence can also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acids, polynucleotides or nucleotide sequences can be double-stranded or single-stranded. The nucleic acids, polynucleotides or nucleotide sequences can be fully or partially synthetic or recombinant.

[0170] Although the antibodies, nucleic acids, vectors or cells of the present invention can be effectively used to combat diseases when used alone, additional therapeutic agents can be used in combination with the antibodies, nucleic acids, vectors or cells of the present invention to combat diseases. Therefore, in another embodiment of the present invention, at least one additional or additional therapeutic agent (e.g., other neurodegenerative disease therapeutic drugs) can be administered to the subject. Therefore, the antibody or its antigen-binding fragment, nucleic acid, vector or cell of the present invention and other therapeutic agents (e.g., other neurodegenerative disease therapeutic drugs) can be administered to the subject. Therefore, the composition or pharmaceutical composition of the present invention can include other active agents or therapeutic agents, as well as the antibody or its antigen-binding fragment, nucleic acid, vector and / or cell of the present invention. However, it should be understood that the antibody or its antigen-binding fragment, nucleic acid, vector or cell of the present invention and other therapeutic agents (e.g., other neurodegenerative disease therapeutic drugs) can be administered separately, for example, by separate administration routes. In addition, the antibody or its antigen-binding fragment, nucleic acid, vector or cell of the present invention and at least one other therapeutic agent (e.g., other neurodegenerative disease such drugs) can be administered sequentially or (substantially) simultaneously. They can be administered in the same pharmaceutical preparation or drug, or they can be formulated and administered separately. For sequential administration, the additional therapeutic agent can be administered at least 1 minute, 10 minutes, 1 hour, 6 hours, 12 hours, 1 day, 5 days, 10 days, 2 weeks, 4 weeks, or 6 weeks before or after administration of the antibody or antigen-binding fragment thereof / nucleic acid / vector / cell.

[0171] "Pharmaceutically acceptable" includes formulations that are sterile and pyrogen-free. Suitable pharmaceutical carriers, diluents, and excipients are well known in the pharmaceutical art. A carrier must be "acceptable" in the sense of being compatible with the drug and not deleterious to the recipient thereof. Typically, the carrier will be saline or an infusion medium (alternatively called an infusion solution), which will be sterile and pyrogen-free; however, other acceptable carriers may be used.

[0172] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

[0173] The composition of the present invention can be administered in a single dose or multiple doses. In particular, the composition can be administered in a single dose.

[0174] Antibodies or antigen-binding fragments thereof, nucleic acids, vectors or compositions of the present invention can be administered in the form of a pharmaceutical preparation comprising an active ingredient by any parenteral route. Depending on the condition and the patient to be treated and the route of administration, the composition can be administered in different doses. In any case, the physician will determine the actual dosage that is most suitable for any individual patient, and it will vary with the age, weight and response of the particular patient.

[0175] In human therapy, the antibody or its antigen-binding fragment, nucleic acid or composition of the present invention is usually mixed with a suitable pharmaceutical excipient, diluent or carrier selected according to the expected route of administration and standard pharmaceutical practice. For each of the embodiments, the antibody or its antigen-binding fragment, nucleic acid molecule or composition of the present invention can be administered by a variety of dosage forms. Examples of such dosage forms include but are not limited to reconstructible powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible particles, cachets, inhalants, aerosol inhalers, patches, particle inhalers, implants, long-acting implants, injections (including subcutaneous, intramuscular, intravenous and intradermal, preferably intravenous), infusions and combinations thereof. Typically, the cells of the present invention can be administered in an injection or infusion buffer. Exemplary formulations can be found in, for example, Remington's Pharmaceutical Sciences, 19th edition., Grennaro, A., ed., 1995, which is incorporated herein by reference.

[0176] Antibody of the present invention or its Fab, nucleic acid or compositions can also be used parenterally, for example intravenously, intraarterially, intraperitoneally, intrathecally, intracranially, locally, intramuscularly, through cheek, subcutaneously, through epidermal, epidural, suction, intracardiac, intraventricular, intraocular, intraspinal, through nose, sublingually, transdermal or transmucosal, or they can be used by infusion technique.They are preferably used in the form of sterile aqueous solution, and it can contain other substances, for example enough salt or glucose so that solution and blood are isotonic.If necessary, the aqueous solution should be suitably buffered (preferably pH is 3 to 9).Preparing suitable parenteral preparation under aseptic condition can easily complete by standard pharmaceutical technology well known to those skilled in the art.

[0177] Formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules, bags, and vials.

[0178] In some embodiments, the antibodies or antigen-binding fragments thereof, nucleic acids or compositions of the present invention are used to treat and / or prevent a neurodegenerative disease in a subject, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the antibodies or antigen-binding fragments thereof, nucleic acids or compositions. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis or spinocerebellar ataxia.

[0179] The term "treat" refers to improving, slowing, reducing or reversing the progression or severity of a disease or condition, or improving, slowing, reducing or reversing one or more symptoms or side effects of such a disease or condition. In the present invention, "treat" also refers to a method for obtaining a beneficial or desired clinical result, wherein "beneficial or desired clinical result" includes but is not limited to relief of symptoms, reduction in the extent of a condition or disease, stabilized (i.e., not worsening) disease or condition state, delay or slowing of disease or condition state progression, improvement or alleviation of a disease or condition state, and alleviation of a disease or condition, whether partial or complete, detectable or undetectable.

[0180] The term "prevention" refers to the administration of the antibodies and functional fragments thereof of the present invention to prevent or hinder the development of at least one symptom of a disease or condition. The term also includes treating subjects in remission to prevent or hinder relapse.

[0181] The term "subject" refers to a warm-blooded animal, preferably a mammal (including humans, livestock and farm animals, zoo animals, sports animals or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.), more preferably humans. In one embodiment, the subject can be a "patient," that is, a warm-blooded animal, more preferably a human, who is waiting to receive or is receiving medical care or will be the subject of a medical procedure or disease progression monitoring. In one embodiment, the subject is an adult (e.g., a subject over 18 years of age). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is a male. In another embodiment, the subject is a female.

[0182] The antibodies and antigen-binding fragments thereof of the present invention can further be used in diagnostic methods or as diagnostic imaging ligands. In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention can be labeled with radioactive markers, fluorescent markers, fluorescein-type markers, rhodamine-type markers, phycoerythrin, umbelliferone, lissamine, cyanine, Texas Red, BODIPY (Invitrogen) or its analogs, horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, streptavidin / biotin and avidin / biotin are labeled or modified. Such labeled or modified antibodies or antigen-binding fragments thereof can be used to detect the presence and / or concentration of amyloid protein in samples, respectively, for clinical diagnosis, experimental detection, etc. The experimental techniques used include but are not limited to ELISA, Dot-blot, Western-blot, chemiluminescence, electrochemiluminescence, Simoa technology, radioactive technology, etc. In some embodiments, paramagnetic labels can also be used, and preferably positron emission tomography (PET) or single photon emission computed tomography (SPECT) is used for detection. Such paramagnetic labels include, but are not limited to, materials containing aluminum (Al), barium (Ba), calcium (Ca), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (M), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), platinum (Pt), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W), and zirconium (Zi), and in particular Co. +2 , CR +2 Cr +3 、Cu +2 、Fe +2 、Fe +3 、Ga +3 、Mn +3 、Ni +2 、Ti +3、V +3 and V +4 Compounds of paramagnetic ions, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions.

[0183] In one embodiment of the present invention, the sample is a biological sample. Examples of biological samples include, but are not limited to, tissue lysates and extracts prepared from diseased tissues, body fluids, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph fluid, ascites, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, and alveolar macrophages.

[0184] In one embodiment of the present invention, the term "sample" is intended to mean a sample taken from an individual prior to any analysis.

[0185] The technical solutions of the present invention will be described in detail below by way of examples, which are descriptive and illustrative only and are not intended to be limiting. The reagents used in the following examples, unless otherwise noted, can be readily purchased from reagent companies such as Sigma Aldrich and Merck. The test methods, unless otherwise noted, can be found in textbooks such as Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.

[0186] Example

[0187] Example 1 Affinity maturation of W20 antibody

[0188] Dilute Aβ oligomers (preparation method, see CN101463082A) to 10-100 μg / mL in coating buffer (PBS, pH 7.4). Add 4 mL to an immunotube and coat overnight at 4°C. Discard the supernatant and quickly rinse the tube three times with PBS. Fill the immunotube with 3% bovine serum albumin (BSA) and block vertically at room temperature for 2 hours. Discard the supernatant and quickly rinse the tube three times with PBS. Suspend the phage antibody library (W20 random mutation library, constructed by introducing random mutations in the antibody CDR regions using error-prone PCR and efficiently transforming Escherichia coli) in 4 mL of 3% BSA and add to the immunotube. Incubate upside down at room temperature for 1 hour, then vertically for 1 hour. Wash 10 times with PBS containing 0.1% Tween-20 (20 washes for the second round of screening and thereafter). After absorbing PBS, add 500 μL of glycine solution (0.1 M, pH = 7.4) to elute the phages and incubate them at room temperature for 10 minutes. 600=0.4 E. coli TG1 (purchased from the UK MRC Center) was added with 250 μL of the eluted phages and incubated at 37°C for 30 min. The remaining eluted phages were stored at 4°C. 10 μL of TG1 culture medium was taken. 4 , 10 6 , 10 8 Spread the 1 / 2 dilution onto 2xTY plates and incubate at 37°C overnight. Centrifuge the remaining TG1 culture at 11,600 g for 5 minutes at 4°C. Resuspend the pellet in 100 μL of culture medium, spread onto 2xTY plates, and incubate at 37°C overnight. Count the colonies growing on the 2xTY plates and calculate the input-output ratio (input-output ratio = phage antibody titer after enrichment / phage antibody titer before enrichment).

[0189] Add 2 mL of 2xTY medium to the plate full of bacterial colonies, scrape the bacteria with a glass rod, collect the bacterial suspension, take 50 μL and add it to 50 mL of 2xTY medium, add ampicillin (Sigma, product number A9518) with a final concentration of 100 μg / mL and 1% glucose to the medium, and shake at 37°C until the OD 600 =0.4. Add 15% glycerol to the remaining bacterial solution and store at -70°C. Add 1x10 11 Helper phage M13K07 (purchased from the MRC Center in the UK) was incubated at 37°C for 30 minutes, and the culture was centrifuged at 3000g for 10 minutes at 4°C. The pellet was resuspended in 50 mL of 2xTY medium, supplemented with Amp (100 μg / mL), 50 μg / mL kanamycin (Kana), and 0.1% glucose to a final concentration of 100 μg / mL, and incubated on a shaker at 30°C overnight. The overnight culture was centrifuged at 3300g for 15 minutes at 4°C, and the supernatant was collected. A 1 / 4 volume of a 20% PEG (polyethylene glycol, PEG-6000) and 2.5 M NaCl solution was added, mixed thoroughly, and incubated at 4°C for at least 1 hour. The cell culture was then centrifuged at 3300g for 30 minutes at 4°C, the supernatant discarded, and the pellet resuspended in 2 mL of PBS. The supernatant obtained by centrifugation at 11600g for 10 minutes at 4°C was the phage antibody display library after the first round of enrichment. After each screening session, 1 μL of phage supernatant was titered. Specifically, the phage stock solution was serially diluted, infected with E. coli at 37°C for 30 minutes, plated onto 2xTY plates, and incubated at 37°C overnight. The colonies growing on the 2xTY plates were counted, and the number of phage clones per unit volume of phage stock solution was calculated using the dilution ratio. This enrichment and screening process was repeated three times, resulting in the second, third, and fourth rounds of screening.

[0190] Table 5 Screening of phage antibody library against Aβ oligomers AβO*W20

[0191] The results, as shown in Table 5, show that the input-output ratio of phage antibodies gradually increased over four rounds of screening. The input-output ratio in the fourth round was over 200 times that of the first round, indicating that phage displaying high-affinity antibodies against Aβ oligomers were effectively enriched. Polyclonal ELISA analysis was also performed on the antibody libraries from the four rounds of screening. Specifically, the binding ability of the phage libraries obtained in each round to Aβ oligomers was determined by ELISA. The experimental principle was to coat Aβ oligomers onto an ELISA plate, then add the phage library (1E10 cfu) to bind to them. The binding was then detected by indirect ELISA using horseradish peroxidase (HRP)-conjugated anti-M13 phage antibodies. The results are shown in Figure 1. The M13 phage group serves as a negative control, and the PBS group serves as a blank control. As can be seen from the figure, the binding ability of the phage antibody library to Aβ oligomers gradually increased from the first round to the fourth round, and was higher than that of the negative control group.

[0192] Example 2 ELISA identification of single-chain antibody monoclonal

[0193] The phages that had undergone the above four rounds of enrichment screening were infected with Escherichia coli HB2151 (purchased from the MRC Center in the UK) and incubated on plates. Single clones were picked and placed on 96-well cell culture plates. 200 μL of 2xTY medium containing 100 μg / mL Amp and 1% glucose was added to each well. Culture was carried out on a 37°C shaker (300 rpm) overnight. 2 μL of bacterial solution was aspirated from each well and added to another new 96-well bacterial culture plate. 200 μL of 2xTY medium containing 100 μg / mL Amp and 1% glucose was added to each well. Culture was carried out on a 37°C shaker. The OD 600After reaching 1.0, glycerol was added to the first plate to a final concentration of 15% and stored at -70°C. IPTG was added to each well of the second 96-well plate to a final concentration of 1 mmol / L and cultured overnight on a shaker at 30°C. Through ELISA analysis of more than 300 monoclonal clones, 6 dominant mutants (positive clones) were selected for gradient ELISA. Specifically, the binding ability of monoclonal phage (1E10 cfu) to Aβ oligomers was determined by ELISA. The experimental principle is to coat Aβ oligomers on the ELISA plate, then add monoclonal phage to bind to it, and then detect and analyze it by indirect ELISA using HRP-conjugated anti-M13 phage antibody. Specifically, 4 gradient dilutions were selected for the 6 dominant mutants, namely 1:10, 1:20, 1:40 and 1:80, respectively. W20 (1:10) was used as a control. The experiment was repeated three times and the average value was taken. The results are shown in Figure 2. It can be seen that the affinity of the six dominant mutants has been greatly improved, especially 3F, which is still higher than W20 diluted 1:10 at a dilution of 1:80, and its affinity is significantly improved.

[0194] Example 3 Single-chain antibody sequence determination

[0195] The positive clones were sequenced and analyzed using the sequencing primers LMB3: 5'CAGGAAACAGCTATGAC 3' (SEQ ID No. 2) and pHEN seq: 5'CTATGCGGCCCCATTCA 3' (SEQ ID No. 3). Clones that matched the basic structure of the antibodies in the antibody library were considered complete single-chain engineered antibodies. The sequences obtained are shown in SEQ ID Nos. 4-9, respectively. The heavy and light chain CDR sequences of antibody 3F are shown in SEQ ID Nos. 10-15, respectively.

[0196] Example 4 Single-chain antibody sequence alignment

[0197] The sequences of the above positive clones were aligned with W20 using ClustalW software, and the results are shown in Figure 20. The antibody amino acid number is the position of the single-chain antibody where the amino acid is located. The light chain CDR3 region corresponds to the Kabat numbering system as follows: (223, L89), (224, L90), (225, L91), (226, L92), (227, L93), (228, L94), (229, L95), (230, L96), (231, L97). P228 in W20 was mutated to R228 in all mutant sequences, indicating that amino acid position 228 is crucial for the binding of the single-chain antibody to Aβ oligomers (amino acid numbering uses the Kabat numbering system, the same below). By comparing the sequences of 13A and 3F, it was found that the affinity of the single-chain antibody was further improved after replacing G226S227 with A226V227. Because glycine at position 226 has similar properties to alanine, this suggests that amino acid position 227, located in the light chain CDR3, is a key site for binding of this single-chain antibody to Aβ oligomers. Furthermore, a comparison of the sequences of 6A, 8H, and 3A revealed that mutation of V226 to more hydrophobic residues, such as W226 or F226, further enhanced its affinity for Aβ oligomers. These results suggest that amino acid position 226, located in the light chain CDR3 of the single-chain antibody sequence, plays a crucial role in the binding of this single-chain antibody to Aβ oligomers. These results indicate that the (N / Q)224-(S / T)225-X226-X227-X228 (SEQ ID No. 16) fragment in the antibody light chain CDR3 sequence (wherein, X226 is any one of glycine, valine, leucine, isoleucine, phenylalanine, tryptophan, or proline; X227 is any one of phenylalanine, tryptophan, tyrosine, alanine, leucine, or isoleucine; and X228 is any one of alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, glycine, serine, threonine, cysteine, asparagine, or glutamine) is the key site for binding of the single-chain antibody to Aβ oligomers.

[0198] Example 5 Molecular simulation of single-chain antibody structure

[0199] This example is based on the single-chain antibody protein sequence, and the original pdb file is established by homology modeling. On the basis of the original pdb file, water and Cl2 were added to adjust the pH value. - and Na +, run the molecular dynamics simulation program to obtain the pdb file at room temperature (298K); based on the pdb file at room temperature, the molecular dynamics software Gromacs is used to calculate the all-atom structure of the single-chain antibody protein. By mutating the amino acid residues at positions 226, 227, and 228 of the single-chain antibody W20 to alanine, the single-chain antibody structure is calculated by molecular simulation, and the results are shown in Figure 3. By analyzing the CDR3 region, it was found that after alanine mutation at position 226, the side chain residue orientation of the arginine residue at position 227 of the single-chain antibody changed, while after alanine mutation at position 227, the structure did not change significantly. As can be seen from the conclusion of Example 4, amino acid residues at positions 226 and 227 are key residues for binding to Aβ oligomers. In this embodiment, it is known from the antibody structure analysis that the single-chain antibody binds to the Aβ oligomer through the amino acid residue at position 227, and the amino acid residue at position 226 can affect the orientation of the amino acid residue at position 227, thus having an important influence on the binding. After alanine mutation at position 228, the structure of the single-chain antibody changed significantly, indicating that the amino acid residue at position 228 plays an important role in maintaining the structure of the single-chain antibody.

[0200] Example 6 Specific Binding of 3F Antibody to Aβ Oligomers

[0201] Dot blot analysis was used to assess antibody binding to Aβ42 monomers and Aβ oligomers. Aβ samples incubated for 0, 0.5, 1, 2, and 120 minutes were spotted onto nitrocellulose membranes and blocked with 5% skim milk for 1 hour at room temperature. The membranes were incubated with detection antibodies 3F or 4G8 (Biolegend, Catalog No. 800704) for 1-2 hours at room temperature, then washed three times with 0.1% TBST for 5 minutes each. HRP-conjugated anti-c-myc secondary antibodies (Santa Cruz, Catalog No. H2317) or goat anti-mouse secondary antibodies (Abcam, Catalog No. ab6789) were added and incubated for 1 hour at room temperature. After three washes with 0.1% TBST for 5 minutes each, the membranes were coated with ECL chemiluminescence solution and analyzed using an Amersham Imager 680 system. The results showed that 3F antibody did not bind to Aβ monomers but primarily bound to Aβ oligomers (Figure 4).

[0202] Example 7 Antibody Affinity Constant K D Determination of

[0203] Surface plasmon resonance (SPR) was used to characterize the K DThe assay was performed. 3F was fixed on a detection chip (CM5) (purchased from Biacore, Sweden) and equilibrated overnight with HBS-EP buffer. Aβ oligomers were diluted with HBS-EP buffer to different concentration gradients. The sample volume was 35 μL, the flow rate was 5 μL / min, the data lag time after sample loading was 120 seconds, and the protein binding signal was detected. Finally, the data was read using BIAcore analysis software and the K of each antibody was calculated. D The affinity constant between 3F and Aβ oligomers was measured to be K D =8.47×l0 -10 M.

[0204] Example 8 3F antibody can significantly inhibit Aβ aggregation and Aβ oligomer-induced cytotoxicity

[0205] According to the method described by Liu et al. (J Nanobiotechnol (2020) 18:160.), the effect of 3F antibody on Aβ aggregation was evaluated by ThT fluorescence method, and the effect of 3F antibody on Aβ oligomer-induced cytotoxicity was evaluated by MTT method. ThT fluorescence results showed that both 3F and W20 antibodies could significantly reduce Aβ aggregation, while Aβ alone continued to aggregate (Figure 5, Panel A). The results of the cell MTT experiment showed that 3F could significantly inhibit the cytotoxicity of SH-SY5Y cells induced by Aβ oligomers, and its inhibitory activity was better than that of W20 antibody (Figure 5, Panel B).

[0206] Example 9 3F antibody can significantly improve the cognitive function of AD transgenic mice

[0207] Six-month-old male APP / PS1 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into three groups of eight each. Each group was administered intranasally with 3F, W20, or PBS (AD CON.) once daily for 28 days. Wild-type control mice were also administered an equal amount of PBS intranasally. Spatial memory and cognitive abilities were assessed using the water maze and Y-maze methods described by Yu et al. (Br J Pharmacol. 2020;177:2860–2871). The results showed that compared with the AD CON. group, APP / PS1 mice treated with 3F significantly shortened the time it took to reach the platform during the training phase (Figure 6, Panel A). During the exploration phase, when the platform was removed, APP / PS1 mice in the 3F group exhibited significant spatially oriented swimming behavior, with a significant increase in the number of platform crossings (Figure 6, Panel B). Similarly, in the Y-maze test, mice in the AD CON. group showed no significant preference for the novel arm. However, APP / PS1 mice in the 3F group spent significantly more time in the novel arm (Figure 6, Panel C). This indicates that 3F antibody can significantly improve the cognitive function of AD transgenic mice. Moreover, compared with W20, 3F has a more significant ability to improve the cognition of AD transgenic mice.

[0208] Example 10 3F antibody can significantly reduce Aβ levels in the brains of AD transgenic mice

[0209] Using 4G8 antibody as the primary antibody, immunohistochemistry was used to detect the level of Aβ plaques in the brains of APP / PS1 mice. Compared with the mice in the AD CON. group, the plaque area in the cortex and hippocampus of the APP / PS1 mice treated with 3F was significantly reduced (Figure 7). ELISA was further used to detect the content of Aβ40 / 42 in the mouse brain homogenate. The results showed that 3F could significantly reduce the levels of Aβ40 and Aβ42 in the brains of APP / PS1 mice (Figure 8). Moreover, compared with W20, 3F could more significantly reduce the levels of plaques and Aβ40 / Aβ42 in the brains of APP / PS1 mice.

[0210] Example 11 3F antibody can significantly reduce the degree of glial cell activation and inflammatory factor levels in the brain of AD transgenic mice

[0211] The main characteristics of neuroinflammation include excessive activation of glial cells and massive release of inflammatory factors. The inventors detected the activation of microglia and astrocytes in the mouse brain by Iba-1 immunostaining and GFAP immunostaining, respectively. The results showed that compared with the AD CON. group of mice, the Iba-1 and GFAP positive staining areas in the cortex and hippocampus of the APP / PS1 mice treated with 3F were significantly reduced (Figure 9), indicating that the 3F antibody can significantly reduce the activation of glial cells in the brain of AD transgenic mice. Further, ELISA was used to determine the content of IL-1β and IL-6 in mouse brain homogenates. Compared with the AD CON. group of mice, the levels of inflammatory cytokines in the brain of APP / PS1 mice treated with 3F were significantly reduced (Figure 10). Moreover, compared with W20, 3F has the ability to more significantly reduce the level of neuroinflammation in the brain of AD transgenic mice.

[0212] Example 12 3F antibody can significantly improve behavioral coordination and cognitive function in PD transgenic mice

[0213] Twelve-month-old male A53T α-synuclein transgenic mice (purchased from Jackson Lab) were randomly divided into three groups of eight each. Each group received intranasal administration of 3F, W20, or PBS (PD CON) once daily for 28 days. Wild-type control mice received an equal volume of PBS intranasally. Motor coordination was assessed using a pole climbing test.

[0214] Pole climbing test: The pole used is a wooden pole with a rough surface and a base, 1 cm in diameter and 50 cm in length. Place the pole steadily in the mouse cage, and place the mouse on top of the pole with its head facing up, so that it automatically turns its head and then climbs down the pole into the cage with its head down. Record the time it takes for the mouse to turn its head and climb into the cage. If the mouse falls, slips, or fails to complete the task, the time it takes to turn its head is recorded as 30 seconds, and the time it takes to climb back into the cage is recorded as 60 seconds. This is done five times a day, with the first two days being the training period and the third day being the test period. The time it takes for each group of mice to turn their heads and climb into the cage during the test period was statistically analyzed.

[0215] The results showed that the time taken by 3F-treated A53T α-synuclein transgenic mice to turn their heads and climb into the cage was significantly shorter than that of the PD CON group. Furthermore, the time taken by the 3F group was even shorter than that of the W20 group (Figure 11, A and B).

[0216] The novel object recognition experiment, designed based on the mouse's natural tendency to explore new objects, consists of a 40 cm × 40 cm × 40 cm white box. The experiment consists of three phases: acclimation, training, and testing. During the acclimation phase, mice were placed in an empty box and allowed to acclimate spontaneously for 5 minutes. After 24 hours, the training phase began, in which two identical objects were placed in the box and allowed to familiarize the mice for 5 minutes. After a 6-hour interval, the testing phase began, in which one of the old objects in the box was replaced with a new one, and the mice were allowed to freely explore the box for 5 minutes. The number of times the mice explored the new and old objects was recorded. The cognitive index was calculated using the following formula: (number of times the new object was encountered – number of times the old object was encountered) / (number of times the new object was encountered + number of times the old object was encountered). To prevent the influence of mouse odor, the box was wiped with 75% alcohol after each mouse's exploration.

[0217] The results showed that the 3F-treated A53T α-synuclein transgenic mice had significantly higher novel recognition abilities than the PD CON group. Furthermore, the 3F group also showed a stronger novel recognition ability than the W20 group (Figure 11, Panel C).

[0218] Example 13 3F antibody can significantly reduce the level of α-synuclein in the brain of PD transgenic mice

[0219] Phosphorylated Ser129α-synuclein (pSer129-α-syn) is a specific pathological form of α-synuclein in the brain of PD patients. The inventors used pSer129-α-syn antibody (Abcam, catalog number ab59264) as a primary antibody and used immunohistochemistry to detect the level of pathological α-synuclein in the brain of A53Tα-synuclein transgenic mice. There were obvious pSer129-α-syn positive areas in the brainstem of PD CON. group mice, while the pSer129-α-syn positive area in the brainstem of A53Tα-synuclein transgenic mice treated with 3F was significantly reduced (Figure 12). Moreover, compared with W20, 3F was able to more significantly reduce the level of pSer129-α-syn in the brainstem of A53Tα-synuclein transgenic mice.

[0220] Example 14 3F antibody can significantly increase the level of tyrosine hydroxylase in the brain of PD transgenic mice

[0221] Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis and plays an important role in dopamine signaling. The expression of TH is negatively correlated with the severity of PD. The inventors used immunohistochemistry to detect the TH level in the brain of A53T α-synuclein transgenic mice. The results showed that compared with WT mice, the expression level of TH in the brainstem of A53T α-synuclein transgenic mice was significantly reduced, while the expression of TH in the brainstem of PD mice after treatment with 3F antibody was significantly increased (Figure 13). Compared with W20, 3F can more significantly increase the TH level in the brain of PD transgenic mice.

[0222] Example 15 3F antibody can significantly improve the spontaneous activity and anxiety behavior of HD transgenic mice

[0223] Five-week-old male R6 / 2 mice (purchased from Jackson Lab) were randomly divided into three groups of eight each. Each group received intranasal administration of 3F, W20, or PBS (PD CON) once daily for 28 days. Wild-type control mice received an equal volume of PBS intranasally. The mice's motor coordination ability was assessed using the open field test.

[0224] The open field test is a method for evaluating the autonomous behavior, exploratory behavior, and stress levels of experimental animals in a novel environment. The open field consists of an open white plastic box (27 cm × 27 cm × 20.3 cm) divided into nine equal areas arranged in a nine-square grid. A camera is mounted above the box to capture the movement of the mice. Mice are placed in the open field one at a time. After acclimation for 1 minute, their movements are recorded by camera for 30 minutes. The inner walls of the box are cleaned with 70% ethanol before each experiment. Three indicators are quantitatively analyzed: total distance traveled, number of stands in all areas, and time spent in the central area.

[0225] The experimental results showed that compared with WT mice, mice in the HD CON group showed a significant decrease in locomotor activity and obvious anxiety symptoms, including a decrease in total distance traveled in the open field, number of hindlimb rearing movements, and time spent in the central area. Treatment with the 3F antibody significantly increased the total distance traveled, number of rearing movements, and time spent in the central area in R6 / 2 mice (Figure 14). Furthermore, compared with W20, 3F significantly improved the locomotor activity and alleviated anxiety in R6 / 2 mice.

[0226] Example 16 3F antibody can significantly reduce the level of mHTT aggregates in the brain of HD transgenic mice

[0227] In the brains of HD patients or HD transgenic animals, the accumulation and deposition of mHTT protein in the nuclei and cytoplasm of neurons is a key pathological feature. The EM48 antibody (Merck Millipore, Cat. No. MAB5374) can specifically identify pathological mHTT aggregates. Immunofluorescence staining of R6 / 2 mouse brain tissue using the EM48 antibody revealed significant EM48-positive staining in the striatum and cortex of HD CON mice. After 3F treatment, the EM48-positive area in the R6 / 2 mouse brains was significantly reduced (Figure 15), indicating that 3F treatment effectively reduced mHTT levels in the brains of HD transgenic mice, with a superior effect compared to W20.

[0228] Example 17 3F antibody can significantly improve the motor function of ALS transgenic mice

[0229] Eleven-week-old male SOD1-G93A transgenic mice (purchased from the Institute of Model Animals, Nanjing University) were randomly divided into three groups of eight each. Each group was intranasally administered with 3F, W20, or PBS (ALS CON) once daily for 28 days. Wild-type control mice were also intranasally administered with an equal volume of PBS. Motor coordination was assessed using the hanging test and rotarod test.

[0230] Hanging test: Place the mouse on the cage lid. Turn the lid over and place it 50 cm from the cage. Observe for 60 seconds and record the time the mouse remains suspended on the lid. Any time the mouse remains suspended for more than 60 seconds is counted as 60 seconds.

[0231] The results showed that compared with ALS CON mice, SOD1-G93A mice treated with 3F were able to maintain their position in the hanging test significantly longer, indicating that 3F can effectively enhance muscle strength in mice (Figure 16A). Furthermore, 3F was more effective than W20.

[0232] Rotarod test: The rotarod test was conducted for 3 consecutive days, each day consisting of two phases: a training phase and a testing phase. During the training phase, the rotarod rotated at 4 rpm. Mice were trained on the rotarod for 5 minutes, then returned to their cages. The testing phase began 1 hour later. During the testing phase, the rotarod was accelerated from a standstill to 40 rpm over 5 minutes. The duration of the mouse's time on the rotarod was recorded, with 300 seconds defined as the duration of the mouse's time on the rotarod without falling. The test was conducted three times per day, with 30-minute intervals between each test. A total of nine tests were performed on each group of mice over the three days. The duration of each test on the rotarod apparatus was statistically analyzed.

[0233] The results showed that the duration of ALS CON. group mice on the rotarod was significantly lower than that of WT mice, while the duration of SOD1-G93A mice in the 3F treatment group on the rotarod was significantly increased. Moreover, compared with the W20 group mice, the duration of 3F group mice on the rotarod was also longer (Figure 16B).

[0234] Example 18 3F antibody can significantly reduce the level of SOD1 aggregates in the brainstem of ALS transgenic mice

[0235] Extensive accumulation of mutant SOD1 aggregates is present in the brainstems of ALS patients and transgenic mice. The present inventors performed SOD1 immunostaining on brain sections of SOD1-G93A mice. The results showed that compared with ALS CON mice, 3F-treated SOD1-G93A mice showed significantly fewer SOD1 aggregates in the brainstems ( FIG17 ). Furthermore, compared with W20, 3F was more effective in reducing SOD1 aggregate levels in the brainstems of ALS transgenic mice.

[0236] Example 19 3F antibody can significantly reduce the activation of glial cells in the brain of ALS transgenic mice

[0237] The inventors assessed the activation of microglia and astrocytes in the brains of SOD1-G93A mice by immunostaining for Iba-1 and GFAP. The results showed that compared with WT mice, the number of activated glial cells in the brains of ALS CON mice was significantly increased, while treatment with 3F significantly reduced the activation of microglia and astrocytes in the brains of SOD1-G93A mice. Compared with W20, 3F had a superior ability to reduce glial cell activation in the brains of ALS transgenic mice (Figure 18).

[0238] Example 20 Improved antibodies such as 3F can significantly improve cognitive function in AD transgenic mice

[0239] Six-month-old male APP / PS1 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into 8 groups of 8 mice each and administered 3F, W20, 3A, 6A, 8H, 11G, 13A or PBS (AD CON.) intranasally once a day for 28 days; wild-type control mice were administered an equal amount of PBS intranasally. The cognitive ability of mice was tested according to the Y-maze test method described by Yu et al. (Br J Pharmacol. 2020; 177: 2860–2871.). The results showed that the mice in the AD CON. group had no obvious preference for the new arm. However, the time that APP / PS1 mice in each antibody group stayed in the new arm was significantly increased (Figure 19). Among them, the 3F antibody has the most significant ability to improve the cognition of AD transgenic mice.

[0240] Example 21 3FI4 antibody (IgG4 subtype of 3F antibody) specifically binds to Aβ oligomers

[0241] The present invention utilizes conventional methods to transform 3F antibodies into IgG4 subtype antibodies, designated as 3FI4 (3FI4-1, 3FI4-2, 3FI4-3, 3FI4-4, whose light chain sequences are all shown in SEQ ID No. 17, heavy chain sequences are shown in SEQ ID No. 18-21, respectively, light chain constant region sequences are shown in SEQ ID No. 22, and heavy chain constant region sequences are shown in SEQ ID No. 23-26, respectively). The binding ability of the antibodies to Aβ oligomers was determined by ELISA. Specifically, the binding ability of the antibodies to Aβ oligomers was determined by ELISA. Aβ oligomers were coated per well at 4°C overnight. The next day, 100 μL / well of each diluted 3FI4 antibody was added to the coated ELISA plate and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and any residual PBST was patted dry. 100 μL / well of a 1:10,000 diluted goat anti-human HRP secondary antibody was added and incubated at 37°C for 1 hour. The plate was washed three times with PBST to remove any residual PBST. 100 μL / well of TMB colorimetric solution was added and color was developed at 37°C or room temperature. The reaction was terminated with 2 M hydrochloric acid, and the absorbance at OD450 nm was measured with a microplate reader (Figure 21). The results demonstrated that the 3FI4 antibody specifically binds to Aβ oligomers.

[0242] Example 22 Aβ oligomer Aβo*3F that specifically binds to 3F has extremely strong toxicity

[0243] 22.1 Acquisition of Aβ Oligomers Specific to 3F

[0244] 22.1.1 In vitro preparation of Aβo*3F

[0245] Dissolve 1 mg of Aβ42, Aβ40, or other forms of Aβ (Zhongpeptide Biochemical Co., Ltd.) in 1 mL of 100% hexafluoroisopropanol (HFIP), vortex for 5 minutes, sonicate in a water bath for 10 minutes, and then aliquot into EP tubes. Allow to evaporate overnight and store at -20°C. Prior to use, dissolve the HFIP-treated aliquot in 50 mM NaOH to a concentration of 1 mg / mL, vortex for 3-5 minutes, sonicate for 1 minute, and then dilute to 10 μM with pre-chilled PBS. Centrifuge at 21,000 g for 30-40 minutes at 4°C, and discard the precipitate (approximately 5% of the starting volume) to obtain Aβ monomers. Incubate the Aβ monomers at 25°C for 2 days and then incubate them with Protein A magnetic beads cross-linked with 3F antibody at 4°C overnight. The next day, the magnetic beads were washed three times with 0.1% PBST and eluted twice with 20-100 mM glycine (pH 2.0) for 3-5 min. The eluate was neutralized to pH 7 with 1 M Tris to obtain sAβo*3F (Aβos prepared in vitro).

[0246] 22.1.2 Isolation and Preparation of Aβo*3F from APP / PS1 Mouse Brain Homogenate and AD Patient CSF

[0247] To isolate and prepare Aβo*3F-specific Aβo (Aβo*3F), APP / PS1 mouse brain homogenate or AD patient CSF samples (obtained from the First Affiliated Hospital of Zhengzhou University, with written informed consent and approval from the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University) were incubated with Protein A magnetic beads cross-linked with 3F antibody overnight at 4°C. The next day, the beads were washed three times with 0.1% PBST and eluted twice with 20 mM-100 mM glycine (specifically, 20 mM, pH 2.0) for 3 min. The eluate was neutralized to pH 7 with 1 M Tris to obtain Aβo*3F from the brains of APP / PS1 mice (mAβo*3F, Aβo*3F isolated from the brains of AD mice) or Aβo*3F extracted from the CSF of AD patients (hAβo*3F, Aβo*3F isolated from the cerebrospinal fluid of AD patients). The mixture of Aβ aggregates after 3F immunodepletion is called Aβ-ID, which includes sAβ-ID (prepared in vitro), mAβ-ID (isolated and prepared from the brain of APP / PS1 mice), and hAβ-ID (isolated and prepared from the CSF of AD patients), and is used as a control.

[0248] Aβ aggregate mixture Aβ*6E10 was prepared by mixing APP / PS1 mouse brain homogenate or AD patient CSF with Protein G magnetic beads cross-linked with 6E10 antibody, reacting overnight at 4°C, and then eluting twice with 20mM-100mM glycine (specifically, 20mM, pH 2.0) for 3-5 minutes (specifically, 3 minutes). The eluate was neutralized with 1M Tris to pH 7 to obtain Aβo*6E10 from APP / PS1 mouse brain (mAβo*6E10, Aβo*6E10 isolated from AD mouse brain) or Aβo*6E10 extracted from AD patient CSF (hAβo*6E10, Aβo*6E10 isolated from AD patient CSF) (Figure 22). The concentration of Aβ obtained by immunoprecipitation was measured using an Aβ detection kit.

[0249] 22.2 Aβo*3F Molecular Size and Morphology

[0250] 500 μL of Aβo*3F (mAβo*3F) from APP / PS1 mouse brain or 100 μL of SEC marker was loaded onto a Superdex 200 10 / 300GL molecular sieve chromatography column connected to an AKTA pure system. The column was pre-equilibrated with Tris-Gly buffer and eluted at a flow rate of 0.5 mL / min. Comparison with the marker revealed a molecular weight of 588 kDa for mAβo*3F (Figure 23, Panel A). Additionally, 10 μL of mAβo*3F was dropped onto a 200-mesh copper grid and adsorbed for 20 minutes. The grid was then blotted dry with filter paper, negatively stained with 2% uranyl acetate for 30 seconds, blotted dry, and air-dried. The sample was examined under a transmission electron microscope (TEM, Hitachi H7700, Japan) at 120 kV and 100,000× magnification. The results showed mAβo*3F particles with a diameter of approximately 10 nm (Figure 23, Panel A). The molecular weight and distribution of sAβo*3F and Aβo*3F (hAβo*3F) extracted from CSF of AD patients were consistent with the results of mAβo*3F.

[0251] 22.3 Strong Cytotoxicity of Aβo*3F

[0252] MTT assays showed that the half-inhibitory concentration (IC50) of sAβo*3F for N2a cell cytotoxicity was approximately 0.186 nM, while the IC50 of sAβos before immunoprecipitation was approximately 63.26 nM, a 340-fold difference (Figure 23, Panels B and C). MTT assays showed that the IC50 of mAβo*3F for N2a cells was approximately 0.111 nM, and the IC50 for primary neurons was approximately 0.057 nM (Figure 23, Panel D). MTT assays showed that the IC50 of hAβo*3F for primary neurons was approximately 0.076 nM (Figure 23, Panel E). These results indicate that Aβo*3F is a highly neurotoxic oligomer.

[0253] 22.3 Aβo*3F causes a significant decline in cognitive function in mice and severely damages neuronal function in their brains

[0254] Three-month-old C57BL / 6 mice were randomly divided into six groups, each with 6-8 mice, and injected intracerebroventricularly: 2.5 nM (45.5 pg) mAβo*3F, 2.5 nM (45.5 pg) mAβ*6E10, 2.5 nM (45.5 pg) mAβ-ID, 600 nM (10.9 ng) mAβ*6E10, 600 nM (10.9 ng) mAβ-ID, and a control group injected with Tris-Gly solvent. Twenty-four hours after injection, behavioral and cognitive abilities of the mice were assessed, and brain pathological analysis was performed.

[0255] Y-maze results showed that mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 spent significantly less time in the novel arm than mice in the control group. However, mice injected with 2.5 nM mAβ*6E10, 2.5 nM mAβ*ID, and 600 nM mAβ*ID showed no significant differences compared to the control group. This suggests that mAβo*3F, but not mAβ*6E10 or mAβ-ID, causes severe memory impairment in mice at low concentrations. For mAβ*6E10, similar levels of memory impairment only occurred when the concentration was increased to 600 nM (Figure 24, Panel A).

[0256] Mouse brain samples were stained using the FD Fast Golgi Staining Kit. Dendritic spine density was calculated, revealing a significant decrease in dendritic spine density in CA1 neurons of mouse brains treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 compared to the control group. However, 2.5 nM mAβ*6E10, 2.5 nM mAβ-ID, or 600 nM mAβ-ID had no significant effect on dendritic spine density in mouse brain neurons (Figure 24, Panels B and C).

[0257] Example 23 Further Optimization and Activity Determination of 3F Antibody

[0258] The 3F antibody was mutated from amino acid K at position 100 to amino acid R (not counting the MA residue introduced for single-chain antibody expression, representing position 98 in the antibody numbering system; this variant is designated K98R mutant, with the sequence shown in SEQ ID NO: 27). The effect of the K98R mutation on the affinity of this single-chain antibody was investigated. The method used is as follows.

[0259] The above Aβ oligomers were coated onto 96-well plates at 100 ng / well and coated overnight at 4°C. The next day, the plates were blocked with 3% BSA at room temperature for 2 hours, washed twice with PBST, and patted dry for later use. K98R mutant and 3F were diluted, starting from 1 μg / ml, and serially diluted for a total of 14 gradients. The diluted antibody solution was added to the ELISA plate coated with the above Aβ oligomers, and parallel wells were made. The plates were incubated at 37°C for 1 hour, washed three times with PBST, and patted dry. 1:10000 diluted goat anti-human IgG-HRP was added, and the plates were incubated at 37°C for 45 minutes, washed three times with PBST, and patted dry. TMB color development solution was added for about 15 minutes. After the stop solution was stopped, the OD 450 The readings were taken in nm, and the values ​​were analyzed, compared and plotted using GraphPad.

[0260] The results, shown in Figure 25 , indicate that mutation of amino acid position 98 of the single-chain antibody heavy chain from K to R further enhances affinity, suggesting that amino acid position 98 plays a key role in the binding of the single-chain antibody to Aβ oligomers. The present inventors investigated the reasons for this finding and discovered that, when numbered according to IMGT, amino acid position 98 also represents the residue in the heavy chain CDR3 of the 3F antibody (according to IMGT numbering, the heavy and light chain CDR sequences of the 3F antibody are: CDR-H1: GFTFSSYA (SEQ ID NO: 28); CDR-H2: ISNLGLTT (SEQ ID NO: 29); CDR-H3: AKTTSRFDY (SEQ ID NO: 30); CDR-L1: QSISSY (SEQ ID NO: 31); CDR-L2: KAS; CDR-L3: QNSAVRPVT (SEQ ID NO: 32)). The present inventors also investigated the in vivo activity of the K98R variant and found that it further enhanced the activity compared to the 3F antibody (data not shown).

[0261] Equivalent plan

[0262] Although multiple embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily envision various other means and / or structures for achieving the functions described herein and / or obtaining the results and / or one or more advantages described herein, and will consider that each such variation and / or modification is within the scope of the present invention. More broadly, a person skilled in the art will readily understand that all parameters, materials, and settings described herein are intended to be exemplary, and that actual parameters, materials, and / or settings will depend on the specific application in which the teachings of the present invention are used. A person skilled in the art will recognize or be able to determine many equivalents to the specific embodiments of the present invention described herein using only routine experimentation. Therefore, it should be understood that the foregoing embodiments and examples are presented by way of example only, and within the scope of the appended claims and their equivalents, the present invention may be implemented in a manner different from that specifically described and claimed. Any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention if such features, systems, articles, materials, and / or methods do not conflict with each other.

[0263] As used in the present specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present in conjunction in some cases and not in other cases. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated otherwise. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a reference to "A and / or B" may, in one embodiment, mean A but not B (optionally including other elements in addition to B); in another embodiment, mean B but not A (optionally including elements in addition to A); in yet another embodiment, mean both A and B (optionally including other elements); and so on.

[0264] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be understood to be inclusive, i.e., including at least one of a plurality of elements or a list of elements, but also including more than one, and optionally, other unlisted items. Only when a contrary term is explicitly indicated, such as "only one of" or "exactly one of", or when used in a claim, "consisting of..." will refer to exactly one element of a plurality of elements or a list of elements. Generally, when preceded by an exclusive term, such as "either", "one of", "only one of" or "exactly one of", the term "or" as used herein should only be understood to represent an exclusive alternative (i.e., "one or the other but not both"). "Substantially consisting of...", when used in a claim, should have its ordinary meaning in the field of patent law.

[0265] As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements of the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") may mean at least one, optionally including more than one A, with no B present (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one B, with no A present (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0266] In the claims and the foregoing description, all conjunctions such as "comprises," "includes," "with," "having," "contains," "involving," "possessing," etc. are to be understood as open-ended, i.e., meaning including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively.

[0267] The use of ordinal terms in the claims, such as "first," "second," "third," etc. to modify claim elements does not in itself imply any priority, precedence, or order of one claim element relative to another claim element, or the temporal order in which actions in a method are performed, but merely serves as a label to distinguish one claim element having a certain name from another element having the same name (but for ordinal terms) to distinguish claim elements.

Claims

1. An improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, comprising an amino acid substitution at one or more positions selected from amino acid residue positions 226, 227, and 228 relative to the W20 antibody, wherein the amino acid sequence of the W20 antibody is shown in SEQ ID No. 1, and the amino acid positions of the antibody are numbered using the Kabat numbering system.

2. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid residue at position 226 is substituted with a non-polar, hydrophobic amino acid with properties similar to alanine, preferably, substituted with glycine, valine, leucine, isoleucine, phenylalanine, tryptophan or proline, more preferably, substituted with glycine, phenylalanine or tryptophan.

3. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid residue 227 is substituted with a hydrophobic amino acid having properties similar to valine, preferably, substituted with phenylalanine, tryptophan, tyrosine, alanine, leucine or isoleucine, more preferably, substituted with phenylalanine, isoleucine or leucine.

4. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the amino acid residue at position 228 is substituted with an aliphatic amino acid having a structure similar to arginine, preferably, with alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, glycine, serine, threonine, cysteine, asparagine or glutamine, more preferably, with lysine, asparagine or glutamine. 5 . The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to claim 1 , wherein amino acid residues 226-228 are substituted with AVR, GSR, WVR, FER, NFR or VRR, respectively. 6 . The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to claim 1 , wherein the amino acid residues at positions 224 and 225 are substituted with glutamine and threonine, respectively.

7. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is an antigen-binding fragment, preferably, an antigen-binding fragment selected from the group consisting of scFv, F(ab')2, Fab', Fab, Fd, Fv, bispecific antibodies, camel antibodies, CDR and antibody minimal recognition unit (dAb), more preferably, an antigen-binding fragment selected from the group consisting of scFv, F(ab')2, Fab', Fab.

8. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Preferably, the antibody is selected from the group consisting of IgG, IgM, IgA, IgD and IgE and their subtypes. More preferably, the antibody is selected from the group consisting of IgG1-4 subtypes. More preferably, the antibody is of IgG4 subtype.

9. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is a scFv and has an amino acid sequence as shown in any one of SEQ ID Nos. 5, 4 and 6 to 9. 10 . The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to claim 1 , wherein the sequence of CDR3 of the light chain thereof is shown as SEQ ID No.

15.

11. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the amino acid sequence at position 98 of the 3F antibody is substituted with arginine. Preferably, the heavy chain amino acid sequence of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID No. 33 to 36. Preferably, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is a scFv and its amino acid sequence is as shown in SEQ ID No.

27.

12. An isolated nucleic acid molecule selected from the group consisting of: (1) DNA or RNA encoding the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11; (2) A nucleic acid molecule that is completely complementary to the DNA or RNA defined in (1).

13. An expression vector comprising the nucleic acid molecule of claim 12 operatively linked thereto. A host cell comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13.

15. A composition comprising the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13 or the host cell according to claim 14, and one or more pharmaceutically acceptable carriers, diluents or excipients, optionally, the composition further comprising another neurodegenerative disease therapeutic agent, preferably, the another neurodegenerative disease therapeutic agent is selected from acetylcholinesterase inhibitors such as donepezil, galantamine, carbastine, and aspartate receptor antagonists such as memantine.

16. A method for producing the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising the steps of: The host cell of claim 14 is cultured under culture conditions suitable for the expression of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, and optionally, the obtained product is isolated and purified.

17. Use of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, the host cell according to claim 14, or the composition according to claim 15 in the preparation of a medicament for inhibiting Aβ aggregation and / or Aβ oligomer-induced cytotoxicity in a subject, or for treating and / or preventing a neurodegenerative disease in a subject, or for diagnosing whether a subject has a neurodegenerative disease.

18. The improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, the host cell according to claim 14, or the composition according to claim 15, for inhibiting Aβ aggregation and / or Aβ oligomer-induced cytotoxicity in a subject, or for treating and / or preventing a neurodegenerative disease in a subject, or for diagnosing whether a subject has a neurodegenerative disease.

19. A method for inhibiting Aβ aggregation and / or Aβ oligomer-induced cytotoxicity in a subject, or treating and / or preventing a neurodegenerative disease in a subject, or diagnosing whether a subject has a neurodegenerative disease, comprising administering to the subject or the subject's cells a therapeutically effective amount of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, the host cell according to claim 14, or the composition according to claim 15.

20. The use according to claim 17, the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition according to claim 18, the method according to claim 19, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia or spinocerebellar ataxia, preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis.

21. Use of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, the host cell according to claim 14 or the composition according to claim 15 in the preparation of a reagent for diagnosing the presence and / or level of toxic forms of amyloid in a sample from a subject.

22. Use of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, the host cell according to claim 14, or the composition according to claim 15 in the preparation of a medicament for specifically binding to the highly toxic amyloid oligomer Aβo*3F in a subject and inhibiting its neurotoxicity, wherein the highly toxic amyloid oligomer Aβo*3F is separated from an Aβ oligomer mixture by immunoprecipitation with an antibody against 3F, and is typically characterized as a high molecular weight Aβ oligomer with a molecular weight of approximately 588 kDa and a diameter of approximately 10 nm based on size exclusion chromatography (SEC) analysis.

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