Improved antibody that specifically binds to amyloid β protein oligomers

Abstract

The present invention relates to an improved antibody that specifically binds to amyloid β oligomers (AβOs). Specifically, the present invention relates to an improved form of the W20 antibody, which has significantly improved affinity for AβOs compared to the W20 antibody, and can more significantly inhibit Aβ aggregation and AβOs-induced neuronal cytotoxicity, more effectively improving cognitive and memory function in Alzheimer's disease model mice and reducing pathological changes in the mouse brain. The improved antibody specifically binds to oligomers of amyloid β, α-synuclein, mHTT, and SOD1 and inhibits the aggregation and cytotoxicity of each amyloid protein, and has greater potential than the W20 antibody for treating multiple amyloid protein-related diseases, such as Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. This improved antibody can specifically bind to the highly toxic amyloid protein oligomer Aβo*3F and has better diagnostic value for AD. The amino acid sequence of the W20 antibody is shown in SEQ ID No. 1.

Description

[Technical Field]

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

[0002] Alzheimer's disease (AD) is a chronic neurodegenerative disease commonly seen in the elderly. Its clinical symptoms are primarily memory decline, cognitive impairment, and loss of independence. AD is ranked fourth in human mortality after cancer, heart disease, and stroke, and its incidence rate shows a clear age-dependent pattern. As the global population ages, the number of AD patients is increasing year by year. Statistics show that there were approximately 50 million AD patients worldwide in 2018, and this number is expected to reach 130 million by 2050. Medical expenditures for AD are enormous: in 2015, the global cost of treating and caring for AD patients was approximately $818 billion, and this is expected to increase to $2 trillion by 2030. Over the past decade, approximately $110 billion has been invested worldwide in research and development of AD treatments. However, despite this massive investment of human and material resources, no specific treatment has emerged, and more than 100 treatments have failed in clinical trials. The development of drugs to address the pathogenic mechanisms of AD has already become the focus of competitive research by many international pharmaceutical companies and research institutions.

[0003] AD is a neurodegenerative disease characterized by memory loss in the elderly, the formation of senile plaques in the brain, and the formation of intraneuronal tangles due to the aggregation of the amyloid protein Aβ and the microtubule-associated protein tau to form toxic oligomers. Internationally, it is generally believed that the most important factor in the onset and progression of AD is Aβ oligomers, which have the greatest cytotoxicity, rather than Aβ monomers or fibrils. The level of Aβ oligomers in the brains of AD patients is highly correlated with the onset, progression, and severity of AD. Aβ oligomers have a high affinity for neural synapses, inducing the redistribution of important synaptic proteins, inducing excessive activation of synaptic glutamate receptors, and destroying neuronal membranes, resulting in the over-activation of Ca2+ in synapses. 2+This disrupts the stabilization level of Aβ, further increasing intracellular oxidative stress and mitochondrial damage. Aβ oligomers bind to neural synapses and activate the complement system, promoting excessive pruning and phagocytosis by microglia, which disrupts the structure of neural synapses, disrupts synaptic function, and causes synaptic damage and excessive loss, leading to cognitive decline. Therefore, targeting Aβ oligomers to suppress Aβ oligomer levels and the neurotoxicity they induce is an ideal strategy for treating AD pathogenesis. However, therapeutic schemes targeting Aβ monomers, especially immunotherapy targeting Aβ monomers, can result in adverse reactions such as inflammation and synaptic loss, leading to frequent failure in clinical trials. Eliminating Aβ and reducing Aβ oligomer levels through immunotherapy strategies has long been a focus of research in the field of AD treatment. In recent years, dozens of antibodies and vaccines targeting Aβ have entered clinical trials. Examples include the first-generation vaccine AN1792, which targets 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). These drugs have shown good therapeutic effects in animal trials, significantly improving the cognitive level of AD transgenic animals and reducing the amount of senile plaques and other pathological changes in the animal's brain. However, few have passed Phase III clinical trials in AD clinical trials due to poor therapeutic efficacy or the occurrence of serious side effects. In June 2021, the US FDA approved the launch of the antibody Aduhelm (aducanumab, a monoclonal antibody) through the accelerated approval program. Aducanumab can specifically recognize Aβ oligomers and has shown some therapeutic efficacy in clinical trials. However, this antibody can cause adverse reactions such as cerebral edema and inflammation, and the therapeutic efficacy of the antibody is also being debated.

[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 also effective targets for the diagnosis and treatment of these diseases. Different amyloid protein oligomers and fibrils deposit in different regions of the cerebral cortex, damaging neurons in the corresponding regions and causing cognitive and behavioral disorders. Although these amyloid proteins have different primary structures, their aggregated oligomers can form similar spatial conformations and have similar toxic mechanisms. Therefore, therapeutic strategies targeting toxic amyloid protein oligomers are of great significance for the diagnosis and treatment of multiple neurodegenerative diseases. Furthermore, research has revealed that in amyloid-related diseases, various amyloid proteins interact synergistically, and the aggregation of one amyloid protein and the onset and progression of disease promote or induce the aggregation of other amyloid proteins, contributing to the pathological process of the disease. For example, clinical studies have found that in the brains of 50% of AD patients, in addition to the presence of Aβ plaques and tau protein neurofibrillary tangles, Lewy bodies (LBs) due to the aggregation and deposition of α-synuclein are clearly present. These patients have more severe pathological features and cognitive decline than AD patients without LB deposits. Transgenic animal experiments have shown that Aβ, tau, and α-synuclein interact synergistically with each other, promoting their aggregation and deposition and accelerating pathological changes and cognitive impairment in the brains of transgenic mice. Therefore, therapeutic approaches targeting only one amyloid protein may not be sufficient to treat amyloid protein-associated diseases, and formulations that specifically target structural features shared by multiple amyloid protein oligomers and are independent of their primary amino acid structure would be an ideal strategy to treat one or more amyloid protein-associated diseases.

[0005] Since 2005, the inventors have been researching amyloid protein oligomer-specific antibodies, and have screened for and obtained the world's first fully human single-chain antibody W20 (Chinese Patent CN101463082A), which can recognize multiple amyloid protein oligomers. This antibody can bind to oligomers of Aβ, α-synuclein, mHTT, SOD1, and other amyloid protein-related diseases such as AD, PD, HD, and ALS, but does not bind to the monomers or fibrils of each amyloid protein. This antibody inhibits the aggregation and cytotoxicity of each amyloid protein, improves cognitive function and behavioral coordination in disease model mice, and can also 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 remains a strong demand in this field for further enhancement of the druggability of amyloid protein oligomer-specific antibodies, and for antibody drugs that effectively treat and / or prevent neurodegenerative diseases. Summary of the Invention

[0007] One aspect of the present invention relates to an improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, which has amino acid substitutions 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 which is set forth in SEQ ID No. 1, and the amino acid positions of the antibody are numbered according to the Kabat numbering system.

[0008] In some embodiments, the 226th amino acid residue of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is substituted with a non-polar and hydrophobic amino acid similar in properties to alanine, preferably with glycine, valine, leucine, isoleucine, phenylalanine, tryptophan, or proline, and more preferably with glycine, phenylalanine, or tryptophan.

[0009] In some embodiments, the 227th amino acid residue of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is substituted with a hydrophobic amino acid similar in properties to valine, preferably phenylalanine, tryptophan, tyrosine, alanine, leucine, or isoleucine, and more preferably phenylalanine, isoleucine, or leucine.

[0010] In some embodiments, the 228th amino acid residue of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is substituted with an aliphatic amino acid similar in structure to arginine, preferably alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, glycine, serine, threonine, cysteine, asparagine, or glutamine, and more preferably lysine, asparagine, or glutamine.

[0011] In some embodiments, the amino acid residues 226 to 228 of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof are substituted with AVR, GSR, WVR, FER, NFR, or VRR, respectively.

[0012] In some embodiments, the amino acid residues at positions 224 and 225 of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof are substituted with glutamine and threonine, respectively.

[0013] In some embodiments, the improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof can specifically bind to Aβo*3F oligomers.

[0014] In some embodiments, the improved amyloid protein 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, camelid antibody, CDR, and minimal antibody recognition unit (dAb), more preferably an antigen-binding fragment selected from the group consisting of scFv, F(ab')2, Fab', and Fab. In some embodiments, the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is scFv or F(ab')2.

[0015] In some embodiments, the improved amyloid protein 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, IgE, and subtypes thereof; more preferably, the antibody is selected from the group consisting of IgG1-4 subtypes; more preferably, the antibody is an IgG4 subtype. In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in any one of SEQ ID Nos. 18-21, and / or the amino acid sequence of the light chain of the antibody is set forth in SEQ ID No. 17. In some embodiments, the amino acid sequence of the heavy chain constant region of the antibody is set forth in any one of SEQ ID Nos. 23-26, and / or the amino acid sequence of the light chain constant region of the antibody is set forth in SEQ ID No. 22.

[0016] In some embodiments, the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is an scFv, and its amino acid sequence is set forth in any one of SEQ ID Nos. 5, 4, and 6-9.

[0017] In some embodiments, the sequence of the CDR3 of the light chain of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is set forth in SEQ ID No. 15.

[0018] In some embodiments, the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof has a substitution of arginine at position 98 relative to antibody 3F. In some embodiments, the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence set forth in any one of SEQ ID Nos. 33-36 and a light chain amino acid sequence as described above. In some embodiments, the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is an scFv, and its amino acid sequence is set forth in SEQ ID No. 27.

[0019] In some embodiments, the present invention relates to variants of the improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof, which have one or more amino acid substitutions, insertions, or deletions relative to the improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or more amino acid substitutions, insertions, or deletions, while still substantially retaining one, more, or all of the biological activities of the improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the present invention relates to variants of the improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof as described above, which have at least 85% identity with the improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof as described above, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.3%, 99.6% or more identity, while still substantially retaining one, several, or all of the biological activities of the improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof as described above. It will be apparent to those skilled in the art that, since a single amino acid residue is used as the basis for calculating the above identity, the above numerical values are only approximate, and the corresponding percentage of identity can be accurately calculated based on the actual number of amino acid substitutions, insertions, or deletions that occur.

[0020] Another aspect of the present invention relates to an isolated nucleic acid molecule selected from:

[0021] (1) DNA or RNA encoding the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof;

[0022] (2) A nucleic acid molecule that is completely complementary to the DNA or RNA defined in (1).

[0023] A further aspect of the present invention relates to an expression vector comprising the nucleic acid molecules as described above operatively ligated together.

[0024] Another aspect of the invention relates to a host cell comprising a nucleic acid molecule or an expression vector as described above.

[0025] Another aspect of the present invention relates to a composition comprising an improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector or a host cell as described above, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0026] Another aspect of the present invention relates to a method for producing an improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof as described above, said method comprising:

[0027] The method includes culturing the host cells as described above under culture conditions suitable for expression of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, and optionally isolating and purifying the resulting product.

[0028] Another aspect of the present invention relates to a composition comprising the above-mentioned improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell, or the above-mentioned improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof obtained by the above-mentioned method, nucleic acid molecule, expression vector, host cell, and another therapeutic agent for neurodegenerative diseases, and preferably the other therapeutic agent for neurodegenerative diseases is selected from acetylcholinesterase inhibitors such as donepezil, galantamine, and rivastigmine, and aspartate receptor antagonists such as memantine.

[0029] Another aspect of the present invention is the use of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition in the manufacture 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 is suffering from a neurodegenerative disease.

[0030] In some embodiments, the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell, or composition is used to inhibit cytotoxicity induced by Aβ aggregation and / or Aβ oligomers in a subject, or to treat and / or prevent a neurodegenerative disease in a subject, or to diagnose whether a subject is suffering from a neurodegenerative disease.

[0031] Another aspect of the present invention relates to a method 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 is suffering from a neurodegenerative disease, said method comprising the step of administering to the subject or a cell of the subject a therapeutically effective amount of an improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition as described above.

[0032] 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 degeneration, preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.

[0033] Another aspect of the present invention relates to the use of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition in the manufacture of a reagent for diagnosing the presence and / or level of a toxic form of amyloid protein in a sample from a subject.

[0034] Another aspect of the present invention relates to the use of an improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell, or composition as described above in the manufacture of a drug for specifically binding to the highly toxic amyloid protein oligomer Aβo*3F in a subject and suppressing its neurotoxicity, wherein the highly toxic amyloid protein oligomer Aβo*3F has been isolated from a mixture of Aβ oligomers by immunoprecipitation against the 3F antibody, and is typically characterized as an Aβ high-molecular-weight oligomer, with a molecular weight of approximately 588 kDa and a diameter of approximately 10 nm based on analysis by size exclusion chromatography (SEC).

[0035] In some embodiments, the amyloid protein is selected from amyloid β protein, α-synuclein, mHTT, and SOD1.

[0036] In other words, to enhance the druggability of antibodies, the present invention performs maturational mutational modifications on the W20 antibody, with the primary mutation site located in the light chain CDR3 region, e.g., by mutating the QTHRP sequence to the NSAVR sequence. Molecular simulation studies demonstrate that the improved antibodies of the present invention, such as antibody 3F, can recognize specific structures on amyloid protein aggregated oligomers. Furthermore, the improved antibodies, such as antibody 3F, have significantly improved binding affinity to antigen Aβ oligomers and significantly prolonged in vivo half-lives. 3F can inhibit the aggregation and cytotoxicity of multiple amyloid proteins, including Aβ, α-synuclein, and mHTT, in vitro. 20-day intranasal administration at μg-level doses significantly improved memory and reduced brain pathological changes and inflammatory responses in AD transgenic mice. Acute toxicity tests using antibodies at more than 1,000 times the therapeutic dose demonstrated good animal tolerance and no pathological changes in organs or tissues. Therefore, 3F has obvious therapeutic effects, good safety, good stability, and relatively good drug formability.

[0037] More significantly, the Aβ oligomers specifically recognized by this antibody are highly toxic oligomers, the most predominant toxic component in the Aβ oligomer mixture, with potent pathogenic effects and playing an important 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 MCI patients, from whom AD is believed to have originated. Their levels show significant differences in the CSF, blood, and / or brain tissue of the three groups: AD patients, MCI patients, and healthy elderly individuals. Therefore, these toxic oligomers can be accurately distinguished from AD patients, MCI patients, and healthy elderly individuals. These toxic oligomers are also present in AD transgenic mice and are directly related to the development of AD transgenic mice. In the present invention, the highly toxic Aβ oligomer recognized by 3F is called AβO*3F. It can be isolated from a mixture of Aβ oligomers by immunoprecipitation (using 3F antibody). Its typical characteristics are high-molecular-weight Aβ oligomers. Based on size-exclusion chromatography (SEC) analysis, its molecular weight is approximately 588 kDa and its diameter is approximately 10 nm. It has a highly toxic effect on neurons, being more than 200 times more toxic than the mixture of Aβ oligomers. AβO*3F can activate microglia and / or astrocytes to secrete large amounts of proinflammatory cytokines. More importantly, the present invention produces AβO*3F in vitro, and the composition of the produced AβO*3F is identical to that of the product immunoprecipitated from cerebrospinal fluid or plasma of AD patients using 3F antibody, and it has the same physicochemical properties and functions. Although some prior art monoclonal antibodies, such as lecanemab, also target Aβ aggregates, the Aβ aggregates targeted by these antibodies are different from the Aβ aggregates targeted by the improved antibodies of the present invention, such as antibody 3F. The 3F antibody specifically targets Aβo*3F, an oligomer with strong neurotoxicity as described above.

[0038] Specifically, the improved antibodies of the present invention, such as antibody 3F, have the following advantages:

[0039] 1) Compared with the W20 antibody, 3F has significantly improved affinity for the highly toxic amyloid protein oligomer Aβo*3F, and can better inhibit amyloid protein aggregation and cytotoxicity, thereby exhibiting better therapeutic potential.

[0040] 2) Aβo*3F, a highly toxic amyloid protein oligomer that specifically binds to 3F, is present in the cerebrospinal fluid (CSF), blood, and / or brain tissue of patients with AD and mild cognitive impairment (MCI), which is believed to be the origin of AD. Its levels differ significantly between the CSF, blood, and / or brain tissues of three groups: AD patients, MCI patients, and healthy elderly individuals. Aβo*3F is an extremely toxic oligomer and the most dominant toxic component in the Aβ oligomer mixture. It has potent pathogenic effects and plays an important role in the development and progression of AD. Therefore, Aβo*3F can be targeted for diagnosing, preventing, and / or treating MCI and / or AD in subjects. 3F targeting Aβo*3F has good application value in the diagnosis and treatment of AD.

[0041] 3) 3F specifically binds to Aβo*3F, a highly toxic amyloid protein oligomer responsible for pathogenesis, without recognizing monomers or fibrils, and without inducing an autoimmune response. Because amyloid protein monomers, such as Aβ and α-synuclein, have normal physiological functions in vivo, antibodies that bind to these monomers are prone to trigger autoimmune responses and simultaneously reduce the effective concentration of the antibody's target in the body, which is one of the major reasons why many antibodies and vaccine drugs have failed in clinical trials. Furthermore, the majority of the dozens of antibodies and vaccines currently in clinical trials targeting Aβ have poor therapeutic efficacy or even serious adverse reactions. Research has shown that many antibodies target Aβ monomers, and their antigen-antibody complexes can induce inflammatory responses and cause neuronal synapse loss, leading to the failure of Aβ-targeting immunotherapy for AD. However, the 3F antibody overcomes these side effects and achieves good AD treatment efficacy.

[0042] 4) 3F can bind to multiple amyloid proteins with different primary structures, reducing their toxicity and promoting their clearance. When amyloid proteins with different primary structures aggregate, they can form similar structures, for example, rich in beta-sheet structure and capable of binding to thioflavin (ThT) or Congo red, providing the basis for 3F to bind to multiple amyloid protein oligomers. 3F specifically binds to Aβ, α-synuclein, mHTT, and SOD1 oligomers and can inhibit the aggregation and cytotoxicity of each amyloid protein. Compared to W20, 3F has more pronounced therapeutic effects in animal models of AD, PD, HD, and ALS, demonstrating greater potential for application.

[0043] 5) 3F has a small therapeutic dose, a clear therapeutic effect, a clear therapeutic mechanism, high safety, and stable properties.

[0044] 6) Because Aβo*3F is in the form of an oligomer formed by the aggregation of Aβ monomers, any antibody targeting Aβ monomers may bind to Aβo*3F. However, the antibodies of the present invention, such as the 3F antibody, specifically recognize and bind to Aβo*3F, whereas antibodies targeting Aβ monomers in the prior art can also bind to multiple forms of Aβ, such as Aβ monomers, oligomers, and fibrils. This significantly reduces the possibility of binding to the highly neurotoxic oligomer Aβo*3F of the present invention, and accordingly, its action and efficacy are dispersed, resulting in a reduced therapeutic effect for AD. [Brief explanation of the drawings]

[0045] [Figure 1] FIG. 1 shows polyclonal ELISA of a phage antibody library. [Figure 2] FIG. 1 shows phage monoclonal ELISA. [Figure 3] FIG. 1 shows a structural molecular simulation of the single-chain antibody W20 and its key amino acid residues. [Figure 4]1 shows that the 3F antibody specifically binds to Aβ oligomers. The binding ability of the 3F antibody to Aβ42 monomers and aggregates was detected by dot blot experiments. [Figure 5] 5A and 5B show that the 3F antibody significantly inhibits Aβ aggregation and Aβ oligomer-induced cytotoxicity. Figure 5A shows the inhibitory effects of the 3F and W20 antibodies on Aβ aggregation detected by thioflavin T (ThT) experiments; Figure 5B shows the inhibitory effects of the 3F and W20 antibodies on Aβ oligomer-induced neuronal cytotoxicity detected by MTT experiments. [Figure 6] Figure 6 shows that the 3F antibody significantly improves the cognitive function of AD transgenic mice. Figure 6A shows the latency of mice in each group to find the platform during the training period in the water maze experiment; Figure 6B shows the number of times mice passed the platform location during the exploration period after the platform was removed in the water maze experiment; and the time spent in the new arm in the CY maze experiment in each group. [Figure 7] 7 shows that the 3F antibody significantly reduces the level of Aβ plaques in the brains of AD transgenic mice. Fig. 7A: Brain sections of APP / PS1 mice were immunostained with the 4G8 antibody to detect the level of senile plaques in the mouse brain; Fig. 7B: Quantitative statistics of the area of senile plaques in the mouse cortex and hippocampus. [Figure 8] 1 shows that the 3F antibody can significantly reduce Aβ levels in the brains of AD transgenic mice. The Aβ40 / 42 content in the brain homogenates of APP / PS1 mice was detected by ELISA. [Figure 9] 9A and 9B show that the 3F antibody significantly reduces the degree of glial cell activation in the brains of AD transgenic mice. Figure 9A shows the detection of microglia and astrocyte activation in the brains of APP / PS1 mice by Iba-1 immunostaining and GFAP immunostaining; Figure 9B shows the quantitative statistics of Iba-1 and GFAP-positive staining areas in the cortex and hippocampus of the mouse brain. [Figure 10]1 shows that the 3F antibody significantly reduces the levels of inflammatory cytokines in the brains of AD transgenic mice. The contents of IL-1β and IL-6 in the brain homogenates of APP / PS1 mice were detected by ELISA. [Figure 11] Figure 11 shows that the 3F antibody significantly improves the behavioral coordination ability and cognitive function of PD transgenic mice. Figure 11A: The time it takes for mice of each group to turn downward in the pole test; Figure 11B: The time it takes for mice of each group to climb into the cage in the pole test; Figure 11C: The cognitive index of mice of each group in the novel object recognition experiment. [Figure 12] Figure 12 shows that the 3F antibody significantly reduces the level of α-synuclein in the brains of PD transgenic mice. Figure 12A: pSer129-α-syn antibody was used to immunostain brain sections of A53T α-synuclein transgenic mice to detect pathological α-synuclein levels in the mouse brain; Figure 12B: Quantitative statistics of the pSer129-α-syn positive staining area in the brainstem region of mice. [Figure 13] Figure 13 shows that the 3F antibody can significantly increase the level of tyrosine hydroxylase in the brains of PD transgenic mice. Figure 13A: Brain sections of A53T α-synuclein transgenic mice were immunostained with tyrosine hydroxylase (TH) antibody to detect the level of TH in the mouse brain; Figure 13B: Quantitative statistics for the area of TH-positive staining in the brainstem region of mice. [Figure 14] FIG. 1 shows statistical analysis of the total path distance (A), number of rearings in all areas (B), and time spent in the central area (C) of mice in each group in an open field experiment, demonstrating that the 3F antibody can significantly improve spontaneous activity and anxiety-inducing behavior in HD transgenic mice. [Figure 15]Figure 15 shows that the 3F antibody can significantly reduce the level of mHTT aggregates in the brains of HD transgenic mice. Figure 15A: Immunostaining of brain sections of R6 / 2 mice with EM48 antibody was performed to detect the level of mHTT aggregates in the mouse brain; Figure 15B: Quantitative statistics of the EM48-positive staining area in the mouse brain. [Figure 16] Figure 16 shows that the 3F antibody can significantly improve the motor function of ALS transgenic mice. Figure 16A shows the residence time of mice from each group in the suspension experiment; Figure 16B shows the duration of mice from each group on the rotarod in the rotarod experiment. [Figure 17] Figure 17 shows that the 3F antibody can significantly reduce the level of SOD1 aggregates in the brainstem of ALS transgenic mice. Figure 17A: Brain sections of SOD1-G93A mice were immunostained with SOD1 antibody to detect the level of SOD1 aggregates in the mouse brain; Figure 17B: Quantitative statistics for the area of SOD1-positive staining in the mouse brainstem. [Figure 18] Figure 18 shows that the 3F antibody significantly reduces the degree of glial cell activation in the brains of ALS transgenic mice. Figure 18A: Iba-1 immunostaining and GFAP immunostaining were used to detect the activation status of microglia and astrocytes in the brains of SOD1-G93A mice; Figure 18B: Quantitative statistics of the Iba-1 and GFAP-positive staining areas in the mouse brains. [Figure 19] FIG. 10 shows the time spent in a new arm by APP / PS1 mice in each group in a Y-maze experiment, demonstrating that the antibody can improve the cognitive function of AD transgenic mice. [Figure 20] FIG. 1 shows the results of comparing the sequences of positive clones with the sequence of W20. [Figure 21] FIG. 1 shows that the 3FI4 antibody (IgG4 subtype of the A16 antibody) can specifically bind to Aβ oligomers. [Figure 22] FIG. 1 is a schematic diagram showing the production of Aβo*3F, Aβ*6E10, and Aβ-ID by immunoprecipitation. [Figure 23]FIG. 1 shows the molecular weight, morphological characteristics and cytotoxicity of Aβo*3F. Figure 23A: Analysis of the molecular weight of mAβo*3F by size exclusion chromatography (SEC); SEC analysis of mAβo*3F and SEC standards (Superdex 200 10 / 300). SEC standards: 1. thyroglobulin (669 kDa), 2. ferritin (440 kDa), 3. aldolase (158 kDa), 4. conalbumin (75 kDa), 5. ovalbumin (44 kDa), 6. carbonic anhydrase (29 kDa); Figure 23B: IC50 of sAβos toxicity against N2a cells; Figure 23C: IC50 of sAβo*3F toxicity against N2a cells; Figure 23D: IC50 of mAβo*3F toxicity against N2a cells and primary neurons; Figure 23E: IC50 of hAβo*3F toxicity against primary neurons. [Figure 24] Figure 24 shows that Aβo*3F significantly reduces cognitive function and damages neurons in the mouse brain. Figure 24A: duration of each group of mice in the novel arm in the Y-maze experiment; Figure 24B: detection of dendritic spine density of mouse neurons by Golgi staining; Figure 24C: statistical analysis of dendritic spine density in B. [Figure 25] This figure shows that the affinity of the single-chain antibody is further improved after K at position 98 in the heavy chain is mutated to R, indicating that the 98th amino acid plays an important role in the binding process of this single-chain antibody with Aβ oligomers. DETAILED DESCRIPTION OF THE INVENTION

[0046] Definition: Amyloid proteins include β-amyloid protein, microtubule-associated protein tau, α-synuclein, huntingtin, pancreatic islet amyloid protein, superoxide dismutase 1 (SOD1), and TDP-43 protein, and include aggregated forms thereof such as monomers, oligomers, protofibrils, or fibers.

[0047] The toxic form of amyloid protein refers to a form of amyloid protein that plays a negative role in the onset and progression of neurodegenerative diseases, such as oligomeric forms and protofibrillar forms.

[0048] Amyloid protein oligomers refer to non-fibrillar aggregates formed by the aggregation of two or more amyloid protein monomer molecules.

[0049] The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule capable of binding to an epitope of an antigen. Naturally occurring antibodies typically comprise a tetramer and are generally composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the isotypes IgG, IgA, IgM, IgD, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Ig molecules of the same class can also be divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and location of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4 subtypes, and IgA can be divided into IgA1 and IgA2 subtypes. Light chains are divided into κ and λ chains by the constant region. The antibodies of the present invention may have any isotype. The choice of isotype is usually determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant region kappa or lambda can be used. If necessary, the class of the antibody of the present invention can be converted using known methods. For example, the class of an initial IgG antibody of the present invention can be converted to an IgM antibody of the present invention. Note that class conversion techniques can also be used to convert an IgG subclass to another subclass, for example, IgG1 to IgG2. Therefore, the effector function of the antibody of the present invention can be converted by isotype switching, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. If the antibody's complement binding activity, e.g., C1q and / or Fc receptor binding activity, is reduced or eliminated, the antibody can be used for various therapeutic purposes. In some embodiments, the antibody of the present invention is an IgM, IgG1, IgG2, IgG3, or IgG4 antibody. An antibody belongs to a particular isotype if its amino acid sequence is nearly identical to that of another isotype.

[0050] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen-binding site and includes natural and artificial antibodies of various structures, including, but not limited to, complete antibodies and antigen-binding fragments of antibodies.

[0051] A "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen. Each heavy chain of an antibody is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), and the heavy chain constant region is 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 regions can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), Fc receptors, and the first component (C1q) of the classical complement system. The heavy and light chain variable regions contain binding regions that interact with antigens. The VH and VL regions can be further divided into hypervariable regions (HVRs) called "complementarity-determining regions (CDRs)," which are separated by more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDR domains and four FR domains, arranged from the amino terminus to the carboxyl terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0052] The terms "complementarity determining region" or "CDR region" or "CDR" (which may be used interchangeably herein with hypervariable region "HVR") refer to the regions of an antibody variable domain that form highly variable sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to an epitope of an antigen. Herein, the three CDRs of a heavy chain are referred to as HCDR1, HCDR2, and HCDR3, and the three CDRs of a light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0053] It should be noted that the CDR boundaries of the variable regions of the same antibody obtained based on different numbering schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined by different numbering schemes will be different. Therefore, when an antibody is defined using a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but whose so-called CDR boundaries differ from the specific CDR boundaries defined in the present invention by applying a different scheme (e.g., a different numbering scheme rule or combination).

[0054] The terms "monoantibody," "monoclonal antibody," or "monoclonal antibody composition" refer to a preparation of antibody molecules of single molecular composition, derived from a population of essentially homogeneous antibodies; i.e., a population comprising individual antibodies that are identical except for any naturally occurring mutations that may be present. A typical monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. In some embodiments, a monoclonal antibody may be composed of two or more Fab domains, thereby increasing its specificity for two or more targets. The terms "monoclonal antibody" or "monoclonal antibody composition" are not limited to any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).

[0055] The present invention further includes "bispecific antibodies," in which the antibodies of the present invention are part of a bivalent or multivalent bispecific framework targeting one or more epitopes (e.g., the second epitope can include an epitope of an active transport receptor, thereby providing the bispecific antibody with improved cell translocation across biological barriers, such as the blood-brain barrier). Thus, in another embodiment, a monovalent Fab of an antibody of the present invention can be linked to a Fab or scfv targeting another, different protein to produce a bispecific antibody. Bispecific antibodies can have dual functions, e.g., a therapeutic function conferred by the present invention, and a transport function that can bind to a receptor molecule and enhance translocation across biological barriers, such as the blood-brain barrier. The terms "biantibody," "bifunctional antibody," "bispecific antibody," "bispecific antibody," or "BsAb" refer to an antibody that has two distinct antigen-binding sites and can simultaneously bind to two target antigens, exerting its antibody targeting properties while also mediating the function of another specialized functional effector molecule. The mediated specific function effector molecule may be a toxin, enzyme, cytokine, radionuclide, etc., and the two antigen-binding arms of the bispecific antibody may each be derived from Fab, Fv, ScFv, dSFv, etc.

[0056] The term "antigen-binding fragment of an antibody" refers to a fragment, portion, region, or domain of an antibody (which may be obtained, for example, by truncation, recombinantly, synthetically, etc.) that is capable of binding to an epitope. An antigen-binding fragment may comprise one, two, three, four, five, or all six CDR domains of such an antibody, and may exhibit different specificities, affinities, or selectivities while still being capable of binding to the epitope. Preferably, the antigen-binding fragment comprises all six CDR domains of the antibody. An antigen-binding fragment of an antibody may be part of or comprises a single polypeptide chain (e.g., an scFv), or may be part of or comprises two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., a bibody, an Fab fragment, an F(ab')2 fragment, etc.).

[0057] Examples of antigen-binding fragments included in the present invention include: (a) Fab' or Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (b) F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge domain; (c) Fd fragment, which is composed of the VH and CH1 domains; (d) Fv fragment, which is composed of the VL and VH domains of one arm of an antibody; (e) single-chain antibody (single chain Fv, scFv), which is a recombinant protein in which the VH and VL of an antibody are linked by a connecting peptide segment using genetic engineering techniques; (f) dAb fragment (Ward et al., Nature, 341, 544-546 (1989)), which is essentially composed of the VH domain and is also called a domain antibody (Holt et al., Trends Biotechnol., 2i(ll):484-90); (g) camelid or nanobody (Revets et al., Expert Opin Biol. Ther., 5(l):111-24) and (h) isolated complementarity determining regions (CDRs).

[0058] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are single-chain antibodies. In some embodiments, the present invention provides single-chain Fvs (scFvs), in which the heavy and light chains in the Fv of an antibody of the present invention are linked by a flexible peptide linker (typically about 10, 12, 15, or more amino acid residues) to form a single peptide chain. Methods for producing such antibodies are described, for example, in U.S. Pat. No. 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.

[0059] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are chimeric antibodies. The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such an antibody, so long as the desired biological activity is exhibited. The present invention provides variable region antigen-binding sequences derived from human antibodies. Therefore, the chimeric antibodies primarily focused on herein include antibodies having one or more human antigen-binding sequences (e.g., CDRs) and one or more sequences derived from a non-human antibody, such as FR or C region sequences. Note that the chimeric antibody described herein refers to an antibody containing human variable region antigen-binding sequences of one antibody class or subclass and other sequences, such as FR or C region sequences, derived from another antibody class or subclass.

[0060] 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 from another mammalian species, such as a mouse species, have been grafted onto human framework sequences, in which additional framework region modifications can be made.

[0061] In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention are human or fully human antibodies. The term "human antibody" or "fully human antibody" ("humAb" or "HuMab") includes antibodies having variable and constant regions derived from human species-based immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human species-based immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or during gene rearrangement, or by somatic mutation in vivo).

[0062] Mutant antibodies are also included within the scope of the present invention. Therefore, variants of the sequences listed herein 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 these variants are also included within the scope of the present invention. For example, amino acid substitution 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 for antibody production.

[0063] The sequences of such variant antibodies have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences listed herein, where such sequence identity is calculated over the full length of the reference sequence (i.e., the sequence listed herein).

[0064] The amino acid residues herein are numbered according to IMGT (registered trademark) (the international ImMunoGeneTics information system) (registered trademark) or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C., (1991), Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, United States Department of Health and Human Services; Chothia, C. & Lesk, AM, (1987), Canonical Structures For The Hypervariable Domains Of Immunoglobulins., J. Mol. Biol., 196, 901-917. Unless otherwise specified, the amino acid residues herein are numbered according to the Kabat numbering system.

[0065] An antibody or antigen-binding fragment thereof "specifically" binds to a region of another molecule (i.e., an epitope) if it reacts or binds to that epitope more frequently, more rapidly, with a longer duration, and / or with greater affinity than it does to other epitopes. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to at least 10 -7 M, e.g. 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody or antigen-binding fragment thereof binds to amyloid protein, particularly its toxic form, with an affinity of M or greater. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, specific binding to amyloid protein, particularly its toxic form, refers to the antibody or antigen-binding fragment thereof's ability to bind to amyloid protein, particularly its toxic form, with the above-mentioned specificity and / or under such conditions. Suitable methods for determining such binding are known in the art.

[0066] In the context of antibody binding to a designated antigen, the term "binding" typically refers to binding of an antibody to a designated antigen within about 10 -6M or lower K D This means that the molecule binds with an affinity corresponding to K D is at least 10-fold, such as at least 100-fold, at least 1,000-fold lower than the affinity of the antibody's binding to a nonspecific antigen other than the designated antigen or a closely related antigen (e.g., BSA, casein).

[0067] As used herein, the term "k d " (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called value.

[0068] As used herein, the term "k a " (M-1 x sec-1 or 1 / Msec) refers to the association rate constant of a particular antibody-antigen interaction.

[0069] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and k d k a This is obtained by dividing by

[0070] As used herein, the term "K A " (M-1 or 1 / M) refers to the binding equilibrium constant of a particular antibody-antigen interaction, and k a k d This is obtained by dividing by

[0071] The fact that changing a single amino acid in a CDR residue results 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) can be used to systematically identify alternative functional CDR sequences. In a preferred method for obtaining such mutant CDRs, the polynucleotide encoding the CDR is mutated (e.g., by random or site-directed mutagenesis) to generate a CDR with a substituted amino acid residue. The substitution score of this substituted BLOSUM62.iij can be determined by comparing the identity of the relevant residue in the original (functional) CDR sequence with that of the substituted (non-functional) mutant CDR sequence. The BLOSUM system analyzes sequence databases to provide amino acid substitution matrices for comparison of reliability (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 shows the BLOSUM62.iij substitution scores (the higher the score, the more conservative the substitution and the more likely it is that the substitution will not affect function). For example, if the resulting antigen-binding fragment containing the CDR cannot bind to PD-L1, the BLOSUM62.iij substitution score is deemed not sufficiently 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 is 0, and more conservative changes (e.g., aspartic acid, asparagine, glutamine, or lysine) are preferred.

[0072] [Table 1]

[0073] Thus, the present invention contemplates the use of random mutagenesis in identifying improved CDRs. In the context of the present invention, conservative substitutions may be defined by substitutions within one or more of the amino acid categories in the following three tables:

[0074] Types of amino acid residues to be conservatively substituted: [Table 2]

[0075] Alternative conservative amino acid residue substitution types: [Table 3]

[0076] Classification of physical and functional alternatives of amino acid residues: [Table 4]

[0077] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0078] In some embodiments, the hydrophilic amino acids are selected from Arg, Asn, Asp, Gln, Glu, His, Tyr, and Lys.

[0079] Additionally, other amino acid groups can be generated using the principles described, for example, in Creighton, (1984), Proteins: Structure and Molecular Properties, WH Freeman and Company.

[0080] Thus, the sequences of the CDR variants of the included antibodies or antigen-binding fragments thereof can differ from the sequences of the CDRs of the parent antibody by substitutions, such as the substitution of 4, 3, 2 or 1 amino acid residues. According to embodiments of the invention, amino acids in the CDR regions may be substituted with conservative substitutions, as defined in the three tables above.

[0081] "Homology" or "sequence identity" refers to the percentage of identical residues between a polynucleotide or polypeptide sequence variant and a non-variant sequence after aligning the sequences and introducing 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 the polynucleotides or polypeptides described herein.

[0082] Such variant polypeptide sequences have 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity to the sequences set forth herein. In other embodiments, the present invention provides polypeptide fragments comprising contiguous stretches of various lengths of the amino acid sequences disclosed herein. For example, where applicable, the peptide sequences provided herein include 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, and all peptides of intermediate lengths therebetween.

[0083] The antibodies of the present invention may be monoclonal antibodies produced by recombinant DNA.

[0084] In some embodiments, the antibodies of the invention are full-length antibodies, preferably IgG1-4 or IgM antibodies, hi other embodiments, the antibodies of the invention are antibody-antigen binding fragments or single chain antibodies.

[0085] The subunit structures and three-dimensional configurations of the constant regions of various immunoglobulins 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 (often amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and amino-terminal to the hinge region of an immunoglobulin heavy chain molecule. As used herein, the term "CH2 domain" includes a portion of a heavy chain molecule, for example, from about residue 244 to residue 360 of an antibody, using conventional numbering schemes (from residue 244 to residue 360, the Kabat numbering system is used; from residue 231 to residue 340, the EU numbering system is used; see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique because it is not closely paired with other domains. Conversely, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule. The CH3 domain has been reported to extend from the CH2 domain to the C-terminus of the IgG molecule and to comprise approximately 108 residues. As used herein, the term "hinge region" includes the portion connecting the CH1 and CH2 domains of a heavy chain molecule. This hinge region comprises approximately 25 residues and is flexible enough to allow the two N-terminal antigen-binding regions to move independently. The hinge region is divided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0086] In some embodiments, the antibody of the present invention is a monovalent antibody, preferably a monovalent antibody having a deletion in the hinge region, for example, as described in WO2007059782 (incorporated herein by reference in its entirety). Thus, in some embodiments, the antibody is a monovalent antibody, wherein the antibody is constructed by the following method: i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen-specific antibody and a nucleotide sequence encoding the constant CL region of an Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of the Ig are operatively linked, and in the case of the IgG1 subclass, the nucleotide sequence encoding the CL region has already been modified so that, in the presence of polyclonal human IgG or when administered to an animal or human, the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides containing the identical amino acid sequence of the CL region; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of the selected antigen-specific antibody. the nucleotide sequence encoding the VH region of a selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operatively linked; iii) providing a cell expression system for producing a monovalent antibody; and iv) co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii) to produce the monovalent antibody.

[0087] Similarly, in some embodiments, the antibodies of the invention are monovalent antibodies,

[0088] (i) a variable region or an antigen-binding portion of said domain of an antibody of the invention as described herein;

[0089] (ii) a CH region of an immunoglobulin or a domain comprising the CH2 and CH3 domains thereof; Here, this CH region or a domain thereof has already been modified so that the hinge region and (if this immunoglobulin is not of the IgG4 subclass) the domain corresponding to another domain of the CH region (e.g., the CH3 domain) do not contain any amino acid residues that can form disulfide bonds with the same CH region or other covalent or stable non-covalent inter-heavy chain bonds with the same CH region in the presence of polyclonal human IgG.

[0090] In some other embodiments, the heavy chain of the monovalent antibody is modified to delete the entire hinge region.

[0091] In another embodiment, the sequence of the monovalent antibody is modified so that it does not contain any acceptor sites for N-linked glycosylation.

[0092] Antibodies of the present invention can be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, used alone or in combination. Generally, if the amino acid sequence of a desired sequence is known, one of skill in the art can readily produce the antibody using standard techniques for producing polypeptides. For example, these antibodies can be synthesized by known solid-phase methods, preferably using a commercially available peptide synthesizer (e.g., an apparatus manufactured by Applied Biosystems, Foster City, California) according to the manufacturer's instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques known in the art. For example, a DNA sequence encoding the antibody can be incorporated into an expression vector and the vector introduced into a suitable eukaryotic or prokaryotic host for expression of the desired antibody, resulting in the antibody as a DNA expression product, which can then be isolated from the host using known techniques.

[0093] The antibodies and antigen-binding fragments thereof of the present invention can be modified by including any "suitable" number of modified amino acids and / or by coupling with coupling substituents. In such cases, "suitable" is typically determined by the ability to retain at least essentially the selectivity for amyloid proteins, particularly their toxic forms, and / or the specificity for amyloid proteins, particularly their toxic forms, relative to the underivatized parent antibody. The inclusion of one or more modified amino acids can contribute, for example, to an increased serum half-life of the polypeptide, a reduced antigenicity of the polypeptide, or improved storage stability of the polypeptide. The modification of one or more amino acids can be made, for example, co-translationally during recombinant production, or post-translationally (e.g., N-linked glycosylation at NXS / T sequences during expression in mammalian cells), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprenylated (e.g., farnesylated, geranyl-geranylated) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, acylated amino acids with biotin, carboxylated amino acids, phosphorylated amino acids, etc. References for performing amino acid modifications are well known in the art, see, for example, Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. Modified amino acids may be selected from, for example, glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, acylated amino acids with biotin, amino acids conjugated to a lipid moiety, or amino acids conjugated to an organic derivatizing agent.

[0094] The antibodies and antigen-binding fragments thereof of the present invention can also be chemically modified by covalent conjugation to a polymer to increase their circulating half-life. Exemplary polymers and methods for linking them to peptides are described in, e.g., U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546. Exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of about 1,000-40,000 D, e.g., about 2,000-20,000 D, e.g., about 3,000-12,000 D).

[0095] The antibodies and antigen-binding fragments thereof of the present invention further include mutants formed by substituting, deleting, or adding one or more amino acids of the above-described anti-amyloid protein antibodies.

[0096] In another aspect, the present invention relates to expression vectors encoding one or more polypeptide chains of an antibody or antigen-binding fragment thereof of the invention, which can be used to recombinantly produce the antibody or antigen-binding fragment thereof of the invention.

[0097] 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 set of appropriate nucleic acid sequences for expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, nucleic acids encoding antibodies of the invention are contained in naked DNA or RNA vectors, such as linear expression elements (e.g., as described in Sykes and Johnston, Nat Biotech, 12, 355-59 (1997)), compact nucleic acid vectors (e.g., as described in US 6,077,835 and / or WO 00 / 70087), plasmid vectors (e.g., pBR322, pUC19 / 18, or pUC118 / 119), minimal size nucleic acid vectors (e.g., as described in Schakowski et al., Mol Ther, 3, 793-800 (2001)), or precipitated nucleic acid vector constructs, such as CaPO4 precipitated constructs (e.g., as described in 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 uses are well known in the art (see, for example, US Pat. No. 5,589,466 and US Pat. No. 5,973,972).

[0098] In some embodiments, the vector is suitable for expressing the antibody or antigen-binding fragment thereof of the present invention in bacterial cells. Examples of such vectors include BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J. Biol. Chem., 264, 5503-5509 (1989)), and pET vectors (Novagen, Madison, Wisconsin).

[0099] The expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors containing constitutive or inducible promoters (e.g., α-factor, alcohol oxidase, and PGH) (for reviews, see 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 in Enzymol, 153, 516-544 (1987)). 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); Beer, 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).

[0100] In the expression vectors of the present invention, the nucleic acid encoding the antibody of the present invention can contain any suitable promoter, enhancer, and other expression-contributing elements, or a combination thereof. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), an effective poly(A) terminator sequence, an origin of replication for producing the plasmid in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid can also contain an inducible promoter for a constitutive promoter (e.g., CMV IE).

[0101] In another aspect, the present invention relates to recombinant eukaryotic or prokaryotic host cells (e.g., transfectomas) that produce the antibodies or antigen-binding fragments thereof of the invention, or the bispecific molecules of the invention. Exemplary host cells include yeast, bacteria, and mammalian cells (e.g., CHO or HEK cells). For example, in some embodiments, the present invention provides cells that contain a nucleic acid stably integrated into the cellular genome, the genome comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof of the invention. In other embodiments, the present invention provides cells that contain a non-integrated nucleic acid (e.g., a plasmid, cosmid, phagemid, or linear expression element), the nucleic acid comprising a sequence encoding an antibody or antigen-binding fragment thereof of the invention.

[0102] The antibodies and antigen-binding fragments thereof of the present invention can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines, and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (e.g., myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep. 18, 1-13, the contents of which are incorporated herein by reference).

[0103] Antibodies of the invention and culture medium are suitably isolated by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0104] The present invention further relates to compositions comprising, consisting of or consisting essentially of an antibody of the invention.

[0105] As used herein, with respect to a composition, "consisting essentially of" means that at least one antibody of the invention, as described above, is the only biologically active therapeutic agent or reagent in the composition.

[0106] In one embodiment, the composition of the present invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient, diluent or carrier.

[0107] The present invention further relates to a medicament comprising, consisting of or consisting essentially of an antibody of the invention and further comprising a pharmaceutically acceptable excipient, diluent or carrier.

[0108] The term "pharmaceutically acceptable carrier" refers to an excipient that does not produce any adverse, allergic, or other untoward reaction when administered to animals, preferably humans. It includes any and all solvents, dispersion media, coating layers, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. For human administration, formulations should meet sterility, pyrogenicity, general safety and purity standards required by regulatory agencies (e.g., FDA office and EMA).

[0109] In some embodiments, the glycosylation of antibodies of the invention is modified. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. Altering glycosylation can, for example, increase the affinity of an antibody for an antigen or alter the ADCC activity of an 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 to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can improve the affinity of the antibody for an antigen. U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al. (incorporated herein by reference) describe such methods in further detail. Alternatively, antibodies with altered glycosylation types can be produced. 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 may be used as host cells to express a recombinant antibody of the invention in the host cell to produce an antibody with altered glycosylation.

[0110] A recombinant expression vector can be introduced into a host cell to produce a transformed host cell. The terms "transform with," "transfect with," "transformation," "introduction," and "transfection" are intended to include introducing a nucleic acid (e.g., a vector) into a cell by one of many possible techniques known to those of skill in the art. As used herein, the terms "transformed host cell" or "transduced host cell" are further intended to include cells transformed with a recombinant expression vector of the invention. Prokaryotic cells can be transformed with a transforming nucleic acid, for example, via electroporation or calcium chloride. For example, nucleic acids can be introduced into mammalian cells by conventional techniques, such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, liposome transfection, electroporation, or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, and other laboratory textbooks.

[0111] Although the nucleotide sequences defined above are DNA, in alternative embodiments of the present invention, the nucleotide sequences may be RNA. Thus, RNA sequences corresponding to the DNA sequences described herein are included. Those skilled in the art should understand how to derive an RNA sequence encoding the same protein / polypeptide product from the DNA sequences shown above. "T" should be replaced with "U".

[0112] As used herein, the terms "nucleic acid sequence" or "nucleic acid molecule" or "polynucleotide," "polynucleotide sequence," or "nucleotide sequence" refer to a sequence of naturally occurring bases, nucleosides, or nucleotide units formed by sugar-sugar (backbone) linkages. The terms further include modified or substituted sequences containing non-naturally occurring units or portions thereof. The nucleic acids, polynucleotides, or nucleotide sequences of the invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine, uracil, and xanthine and hypoxanthine. The nucleic acid, polynucleotide, or nucleotide sequence may be double-stranded or single-stranded. The nucleic acid, polynucleotide, or nucleotide sequence may be wholly or partially synthetic or recombinant.

[0113] Although the antibodies, nucleic acids, vectors, or cells of the present invention can effectively combat diseases when used alone, they can also be used in combination with other therapeutic agents to combat diseases. Therefore, in other embodiments of the present invention, a subject can be administered at least one other or additional therapeutic agent (e.g., another drug for treating a neurodegenerative disease). Therefore, a subject can be administered an antibody or antigen-binding fragment thereof, nucleic acid, vector, or cell of the present invention and another therapeutic agent (e.g., another drug for treating a neurodegenerative disease). Therefore, a composition or pharmaceutical composition of the present invention can contain another active or therapeutic agent, an antibody or antigen-binding fragment thereof, nucleic acid, vector, and / or cell of the present invention. However, it should be understood that the antibody or antigen-binding fragment thereof, nucleic acid, vector, or cell of the present invention and the other therapeutic agent (e.g., another drug for treating a neurodegenerative disease) can be administered separately, for example, via different administration routes. Furthermore, the antibody or antigen-binding fragment thereof, nucleic acid, vector, or cell of the present invention and at least one other therapeutic agent (e.g., another drug for treating a neurodegenerative disease) can be administered sequentially or (essentially) simultaneously. They can be administered in the same pharmaceutical formulation or drug, or can be prepared and administered separately. In the case of sequential administration, the other therapeutic agent may 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.

[0114] "Pharmaceutically acceptable" includes formulations that are sterile and pyrogen-free. Suitable drug carriers, diluents, and excipients are well known in the art of pharmacy. A carrier must be "acceptable" in the sense of being compatible with the drug and not harmful to the user. Usually, the carrier is saline or an injectable medium (also called an injectable solution), which is sterile and pyrogen-free, although other acceptable carriers may be used.

[0115] The pharmaceutical composition of the present invention may be administered in a manner appropriate 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, and the appropriate dosage may be determined by clinical trials.

[0116] The compositions of the present invention may be administered in a single dose or multiple doses, in particular the compositions may be administered all at once in a single dose.

[0117] The antibodies or antigen-binding fragments thereof, nucleic acids, vectors, or compositions of the present invention may be administered as pharmaceutical formulations containing the active ingredient via any parenteral route. Depending on the disease and patient being treated and the route of administration, the compositions may be administered in different dosages. In any case, the physician will determine the actual dosage most suitable for an individual patient, which will vary depending on the age, weight, and response of the particular patient.

[0118] In human therapeutics, the antibodies or antigen-binding fragments thereof, nucleic acids, or compositions of the present invention are typically administered in admixture with appropriate pharmaceutical excipients, diluents, or carriers selected based on the intended route of administration and standard pharmaceutical practice. In each of the above embodiments, the antibodies or antigen-binding fragments thereof, nucleic acid molecules, or compositions of the present invention may be administered in a variety of dosage forms. Examples of such dosage forms include, but are not limited to, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, powders, pellets, particles, microparticles, dispersible particles, wafers, inhalants, aerosols, patches, particle inhalants, implants, long-acting implants, injections (including subcutaneous, intramuscular, intravenous, and intradermal, preferably intravenous), infusions, and combinations thereof. Typically, the cells of the present invention may be administered in an injection or infusion buffer. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition, Grennaro, A., ed., 1995, incorporated herein by reference.

[0119] The antibodies or antigen-binding fragments thereof, nucleic acids, or compositions of the present invention may be administered parenterally, for example, intravenously, intraarterially, intraperitoneally, intrathecally, intracranially, topically, intramuscularly, buccally, subcutaneously, transepidermally, epidurally, by inhalation, intracardiac, intraventricular, intraocular, intraspinal, intranasal, sublingually, transdermally, or transmucosally, or by infusion techniques. They are preferably used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be suitably buffered (preferably pH 3-9). The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished by standard pharmaceutical techniques known to those skilled in the art.

[0120] The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules, packets, vials, etc.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof, nucleic acid, or composition of the present invention is 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 antibody or antigen-binding fragment thereof, nucleic acid, or composition. 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 degeneration.

[0122] The term "treatment" refers to ameliorating, alleviating, attenuating, or reversing the progression or severity of a disease or condition, or ameliorating, alleviating, attenuating, or reversing one or more symptoms or side effects of such a disease or condition. In the present invention, "treatment" also refers to an approach for obtaining a beneficial or promising clinical result, where "beneficial or promising clinical result" includes, but is not limited to, alleviation of symptoms, reduction in the condition or extent of disease, stabilized (i.e., not worsening) state of the disease or condition, delaying or alleviating the progression of the condition of the disease or condition, improvement or palliation of the condition of the disease or condition, and remission of the disease or condition, whether partial or total, detectable or undetectable.

[0123] The term "prevention" refers to preventing or inhibiting the development of at least one symptom of a disease or condition by administering the antibodies and functional fragments thereof of the present invention. This term further includes treating a subject in remission to prevent or inhibit recurrence.

[0124] The term "subject" refers to a warm-blooded animal, preferably a mammal (human, domestic and farm animals, zoo animals, sport or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc.), more preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting admission, receiving medical care, or the subject of a medical program or disease progression monitoring. In one embodiment, the subject is an adult (e.g., a subject 18 years of age or older). In another embodiment, the subject is a child (e.g., a subject under 18 years of age). In one embodiment, the subject is male. In another embodiment, the subject is female.

[0125] The antibodies and antigen-binding fragments thereof of the present invention may also be used in diagnostic methods or as ligands for diagnostic imaging. In some embodiments, the antibodies and antigen-binding fragments thereof may be labeled or modified with radioactive labels, fluorescent labels, fluorescein-type labels, rhodamine-type labels, phycoerythrin, umbelliferone, Lissamine, cyanine, Texas Red, BODIPY FL-SE® (Invitrogen) or its analogs, horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, streptavidin / biotin, and avidin / biotin. Such labeled or modified antibodies or antigen-binding fragments thereof may be used to detect the presence and / or concentration of amyloid protein in a sample for clinical diagnosis, laboratory detection, etc. Laboratory techniques that may be used include, but are not limited to, ELISA, dot blot, Western blot, chemiluminescence, electrochemiluminescence, Simoa technology, radioactive techniques, etc. In some embodiments, paramagnetic labels may also be used, preferably detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT). Such paramagnetic labels include 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), zirconium (Zi), and especially Co. +2 , C.R. +2 , Cr +3 , Cu +2 , Fe +2 , Fe +3 , Ga +3 , Mn +3 , Ni +2, Ti +3 , V +3 and V +4 These include, but are not limited to, paramagnetic ion compounds containing various positron-emitting metals by positron emission tomography and non-radioactive paramagnetic metal ions.

[0126] In one embodiment of the invention, the sample is a biological sample, examples of which include, but are not limited to, diseased tissues and body fluids, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascites, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid, and tissue hydrolysates and extracts prepared from alveolar macrophages.

[0127] In one embodiment of the invention, the term "sample" refers to a sample taken from an individual prior to any analysis.

[0128] The technical solutions of the present invention will be specifically described below through examples, but these examples are for illustrative or exemplary purposes only and are not limiting. Unless otherwise noted, all reagents used in the following examples can be easily purchased from reagent companies such as Sigma-Aldrich and Merck. Unless otherwise noted, the test methods can be found in textbooks such as Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York. [Example]

[0129] Example 1 Affinity maturation of W20 antibody

[0130] Aβ oligomers (see CN101463082A for preparation methods) were diluted to 10–100 μg / mL in coating buffer (PBS, pH 7.4), and 4 mL of the solution was placed in an immunotube and coated overnight at 4°C. The supernatant was discarded, and the tube was quickly washed three times with PBS. The immunotube was filled with 3% bovine serum albumin (BSA) and blocked vertically at room temperature for 2 h. The supernatant was discarded, and the tube was quickly washed three times with PBS. A phage antibody library (W20 random mutation library, constructed by efficiently transforming E. coli with random mutations introduced into the CDR regions of antibodies using error-prone PCR) was suspended in 4 mL of 3% BSA and added to the immunotube. The tube was incubated end-over-end at room temperature for 1 h, followed by another 1 h of vertical incubation. The tube was washed 10 times with PBS containing 0.1% Tween-20 (20 times for the second screening and subsequent screening). After the PBS was dried by aspiration, 500 μL of glycine solution (0.1 M, pH 7.4) was added to elute the phages, which were then incubated at room temperature for 10 minutes by inversion. 250 μL of the eluted phages were analyzed by OD 600 The resulting solution was added to 1.75 mL of E. coli TG1 (purchased from the UK MRC Center) with a pH of 0.4, and incubated at 37°C for 30 minutes in a static state. The remaining eluted phage was stored at 4°C. 4 , 10 6 , 10 8 The 2xTY plates were coated with the 2x diluted solutions and incubated overnight at 37°C. The remaining TG1 culture was centrifuged at 11,600 g for 5 minutes at 4°C, and the precipitate was resuspended in 100 μL of medium. The precipitate was then coated onto 2xTY plates and incubated overnight at 37°C. The clones grown on the 2xTY plates were counted, and the input / output ratio was calculated (input / output ratio = phage antibody titer after enrichment / phage antibody titer before enrichment).

[0131] Add 2 mL of 2xTY medium to a plate where the bacterial clones reached confluence, scrape the bacteria with a glass rod, collect the bacterial suspension, and add 50 μL to 50 mL of 2xTY medium. Add ampicillin (Sigma, Cat. No. A9518) to a final concentration of 100 μg / mL and 1% glucose to the medium, and incubate at 37°C until OD 600 The remaining bacterial solution was added with 15% glycerol and stored at -70°C. 10 mL of the culture was diluted with 1x10 11 The helper phage M13K07 (purchased from the UK MRC Centre) was added and incubated stationary at 37°C for 30 min. The bacterial suspension was centrifuged at 3000g for 10 min at 4°C. The pellet was resuspended in 50 mL of 2xTY medium, supplemented with final concentrations of 100 μg / mL ampicillin, 50 μg / mL kanamycin (Kana), and 0.1% glucose, and cultured overnight on a shaker at 30°C. The overnight culture was centrifuged at 3300g for 15 min at 4°C. The supernatant was collected and a 1 / 4 volume of a mixture of 20% PEG (polyethylene glycol, PEG-6000) and 2.5 M NaCl was added. The mixture was thoroughly mixed and left at 4°C for at least 1 hour. After centrifugation at 3300g for 30 min at 4°C, the supernatant was completely discarded and the pellet was resuspended in 2 mL of PBS. The supernatant was centrifuged at 11,600 g for 10 minutes at 4°C. This was the first round of enriched phage antibody display library. After each screening run, 1 μL of phage supernatant was sampled and antibody titers were measured. Specifically, the phage stock solution was serially diluted and then infected with E. coli at 37°C for 30 minutes, then coated onto 2xTY plates and cultured overnight at 37°C. The clones that grew on the 2xTY plates were counted, and the number of phage clones per unit volume of phage stock solution was calculated based on the dilution rate. The above enrichment screening process was repeated three times: the second, third, and fourth rounds of screening.

[0132] Phage antibody library screening against Aβ oligomer AβO*W20 [Table 5]

[0133] The results are shown in Table 5. After four rounds of screening, the input-output ratio of phage antibodies gradually improved. The input-output ratio in the fourth round of screening was over 200-fold higher than in the first round, indicating effective enrichment of phages with high affinity for Aβ oligomers. At the same time, polyclonal ELISA analysis was performed on the antibody libraries obtained in each round of screening. Specifically, the binding ability of the phage library obtained in each round to Aβ oligomers was measured using ELISA. The experimental principle was as follows: Aβ oligomers were coated onto an ELISA plate, and the phage library (1E10 cfu) was added and allowed to bind. Subsequently, detection was performed using horseradish peroxidase (HRP)-conjugated anti-M13 phage antibodies via indirect ELISA. The results are shown in Figure 1. Among them, the M13 phage group was the negative control and the PBS group was the blank control. As can be seen from the figure, from the first phage antibody library to the fourth phage antibody library, the binding ability to Aβ oligomers showed a gradual increase, and was higher than that of the negative control group.

[0134] Example 2 Identification of monoclonal single-chain antibodies by ELISA

[0135] The phages that had passed the four rounds of enrichment screening were infected into E. coli HB2151 (purchased from the UK MRC Center), coated onto plates, and incubated. Monoclonals were then collected and placed in a 96-well cell culture plate. 200 μL of 2xTY medium was added to each well, containing 100 μg / mL ampicillin and 1% glucose. The plates were then cultured overnight at 37°C on a shaker (300 r / min). 2 μL of the bacterial solution per well was then added to another new 96-well bacterial culture plate, and 200 μL of 2xTY medium was added per well, containing 100 μg / mL ampicillin and 1% glucose. The plates were then cultured at 37°C on a shaker until OD was reached. 600After the pH reached 1.0, glycerol was added to the first plate to a final glycerol concentration of 15% and stored at -70°C. IPTG was added to the second 96-well plate to a final concentration of 1 mmol / L per well and incubated overnight at 30°C on a shaker. ELISA analysis of over 300 monoclonal antibodies was performed, and six dominant mutants (positive clones) were selected for stepwise ELISA. Specifically, the binding ability of monoclonal phage (1E10 cfu) to Aβ oligomers was measured using the following experimental principle: Aβ oligomers were coated onto an ELISA plate, and then monoclonal phage was added and allowed to bind. Subsequently, detection and analysis were performed using HRP-conjugated anti-M13 phage antibodies by indirect ELISA. Specifically, the six dominant mutants were diluted in four steps of 1:10, 1:20, 1:40, and 1:80, and the experiment was repeated three times using W20 (1:10) as a control. The average values were calculated and the results are shown in Figure 2. As can be seen, the affinity of all six dominant mutants was significantly improved. In particular, 3F, even at a dilution of 1:80, was still higher than that of W20 diluted at 1:10, demonstrating a significant improvement in affinity.

[0136] Example 3 Determination of the sequence of a single-chain antibody

[0137] 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 matching the basic structure of the antibodies in the antibody library were identified as complete single-chain engineered antibodies. The sequences are shown in SEQ ID Nos. 4-9, respectively, with the heavy and light chain CDR sequences of antibody 3F shown in SEQ ID Nos. 10-15, respectively.

[0138] Example 4 Sequence alignment of single chain antibodies

[0139] The positive clones and W20 were sequence aligned using ClustalW software, and the results are shown in Figure 20. The antibody amino acid numbers indicate the positions of the single-chain antibody where the amino acids are located. The correspondence between the light chain CDR3 region and the Kabat numbering system is as follows: (223, L89), (224, L90), (225, L91), (226, L92), (227, L93), (228, L94), (229, L95), (230, L96), and (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 single-chain antibodies to Aβ oligomers (amino acids are numbered according to the Kabat numbering system, as follows). Sequence alignment of 13A and 3F revealed that the affinity of the single-chain antibody was further improved after G226S227 was replaced with A226V227. Because the 226th amino acid, glycine, has properties similar to alanine, this suggests that the 227th amino acid in the light chain CDR3 is an important site for the binding of the single-chain antibody to Aβ oligomers. Furthermore, sequence alignment of 6A, 8H, and 3A revealed that the affinity for Aβ oligomers was further improved when V226 was mutated to more hydrophobic amino acid residues, such as W226 or F226. These results suggest that the 226th amino acid in the light chain CDR3 of the single-chain antibody plays an important role in the binding process of this single-chain antibody to Aβ oligomers.These results demonstrate 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, and proline; X227 is any one of phenylalanine, tryptophan, tyrosine, alanine, leucine, and isoleucine; and X228 is any one of alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, glycine, serine, threonine, cysteine, asparagine, and glutamine) is an important site for binding between the single-chain antibody and Aβ oligomers.

[0140] Example 5 Molecular simulation of the structure of a single-chain antibody

[0141] In this example, the original pdb file was established by homology modeling based on the protein sequence of a single-chain antibody. Based on the original pdb file, the Cl-containing nucleotides were extracted with water and pH adjusted. - and Na +A molecular dynamics simulation program was run to obtain a human pdb file at room temperature (298 K). Based on the pdb file at room temperature, the full atomic structure of the single-chain antibody protein was calculated using the molecular dynamics software Gromacs. The amino acid residues 226, 227, and 228 of the single-chain antibody W20 were mutated to alanine, and the structure of the single-chain antibody was calculated using molecular simulation. The results are shown in Figure 3. Analysis of the CDR3 region revealed that the orientation of the side chain residue of the arginine residue at position 227 of the single-chain antibody changed after the mutation of position 226 to alanine, but there was no significant change in the structure after the mutation of amino acid 227 to alanine. As can be seen from the conclusions of Example 4, amino acid residues 226 and 227 are important residues for binding to Aβ oligomers. In this example, analysis of the antibody structure revealed that the single-chain antibody bound to Aβ oligomers via amino acid residue 227, and that amino acid residue 226 had a significant effect on this binding because it could affect the orientation of amino acid residue 227. After mutation of amino acid residue 228 to alanine, the structure of the single-chain antibody changed significantly, indicating that amino acid residue 228 plays an important role in maintaining the structure of the single-chain antibody.

[0142] Example 6 Specific binding of 3F antibody to Aβ oligomers

[0143] Dot blot experiments were used to assess antibody binding to Aβ42 monomers and Aβ oligomers. Aβ samples incubated for 0, 0.5, 1, 2, and 120 min were spotted onto nitrocellulose membranes and blocked with 5% skim milk for 1 h at room temperature. The membranes were then incubated with the detection antibodies 3F or 4G8 (Biolegend, catalog no. 800704) for 1–2 h at room temperature, washed three times for 5 min each with 0.1% TBST, and incubated with HRP-conjugated anti-c-myc secondary antibody (Santa Cruz, catalog no. H2317) or goat anti-mouse secondary antibody (Abcam, catalog no. ab6789) for 1 h at room temperature. After washing three times for 5 min each with 0.1% TBST, ECL chemiluminescence solution was applied to the membranes. Analysis using an Amersham Imager 680 imaging system revealed that the 3F antibody did not bind to Aβ monomers but primarily to Aβ oligomers (Figure 4).

[0144] Example 7 Antibody affinity constant K D Measurement of

[0145] Surface plasmon resonance (SPR) was used to detect antibody K. D The 3F was immobilized on a detection chip (CM5) (purchased from Biacore, Sweden) and equilibrated overnight with HBS-EP buffer. Aβ oligomers were diluted to different concentration gradients with HBS-EP buffer. The loading volume at the time of detection was 35 μL, the flow rate was 5 μL / min, the data delay time after loading was 120 seconds, and the protein binding signal was detected. Finally, the data was read using BIAcore's analysis software to determine the K of each antibody. D The affinity constant between 3F and Aβ oligomers was measured as K D =8.47×l0 -10 It was M.

[0146] Example 8: 3F antibody significantly inhibits cytotoxicity induced by Aβ aggregation and Aβ oligomers

[0147] Following the method described by Liu et al. (J Nanobiotechnol (2020) 18:160.), we evaluated the effect of the 3F antibody on Aβ aggregation using the ThT fluorescence method and on Aβ oligomer-induced cytotoxicity using the MTT method. The ThT fluorescence results revealed that both the 3F and W20 antibodies significantly reduced Aβ aggregation, whereas Aβ alone maintained aggregation (Figure 5A). The cellular MTT experiment results revealed that 3F significantly inhibited Aβ oligomer-induced SH-SY5Y cytotoxicity, with its inhibitory activity being superior to that of the W20 antibody (Figure 5B).

[0148] Example 9: 3F antibody can significantly improve cognitive function in AD transgenic mice

[0149] Six-month-old male APP / PS1 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into three groups of eight mice each and administered 3F, W20, or PBS (AD control) intranasally once daily for a total of 28 days. Mice in the wild-type control group received the same amount of PBS intranasally. Spatial memory and cognitive abilities of the mice were assessed using the water maze and Y-maze methods described by Yu et al. (Br J Pharmacol. 2020;177:2860-2871). Results showed that during the training period, APP / PS1 mice treated with 3F significantly reduced the time it took to reach the platform compared with the AD control group (Figure 6A). During the exploration period, when the platform was removed, APP / PS1 mice in the 3F group demonstrated clear spatially oriented swimming behavior, with a significantly increased number of crossings over the platform (Figure 6B). Similarly, in the Y-maze experiment, mice in the AD control group showed no clear preference for the new arm. In contrast, the time spent in the new arm of the APP / PS1 mice in the 3F group was significantly increased (Figure 6C). This indicates that the 3F antibody significantly improved the cognitive function of the AD transgenic mice. Furthermore, 3F significantly improved the cognitive ability of the AD transgenic mice compared to W20.

[0150] Example 10: 3F antibody can significantly reduce Aβ levels in the brains of AD transgenic mice

[0151] The levels of Aβ plaques in the brains of APP / PS1 mice were detected by immunohistochemistry using the 4G8 antibody as the primary antibody. Compared with AD control mice, the plaque area in the cortex and hippocampus of APP / PS1 mice treated with 3F was significantly reduced (Figure 7). Furthermore, the Aβ40 / 42 content in the brain homogenates of the mice was detected by ELISA. The results showed that 3F significantly reduced the levels of Aβ40 and Aβ42 in the brains of APP / PS1 mice (Figure 8). Furthermore, 3F significantly reduced the levels of plaques and Aβ40 / Aβ42 in the brains of APP / PS1 mice compared with W20.

[0152] Example 11: 3F antibody significantly reduces the activation level of glial cells and the level of inflammatory cytokines in the brains of AD transgenic mice

[0153] Key features of neuroinflammation include excessive glial cell activation and the massive release of proinflammatory cytokines. We detected the activation of microglia and astrocytes in the mouse brains using Iba-1 immunostaining and GFAP immunostaining, respectively. The results showed that the areas of Iba-1 and GFAP-positive staining in the cortex and hippocampus of APP / PS1 mice treated with 3F were significantly reduced compared to AD control mice (Figure 9), indicating that the 3F antibody significantly reduced glial cell activation in the brains of AD transgenic mice. Furthermore, the contents of IL-1β and IL-6 in mouse brain homogenates were measured by ELISA. Compared to AD control mice, the levels of inflammatory cytokines in the brains of APP / PS1 mice treated with 3F were significantly reduced (Figure 10). Furthermore, 3F significantly reduced the level of neuroinflammation in the brains of AD transgenic mice compared to W20.

[0154] Example 12: 3F antibody significantly improves behavioral coordination and cognitive function in PD transgenic mice

[0155] Twelve-month-old male A53T α-synuclein transgenic mice (purchased from Jackson Lab) were randomly divided into three groups of eight mice each. 3F, W20, or PBS (PD control) was administered intranasally once daily for a total of 28 days. Mice in the wild-type control group were administered the same amount of PBS intranasally. The mice's motor coordination was assessed using a pole test.

[0156] Pole test: The pole used was a wooden pole with a rough surface and a base, 1 cm in diameter and 50 cm in length. The wooden pole was placed stably inside the mouse cage. The mouse was placed on top of the wooden pole with its head facing up. The mouse automatically rotated downward, then turned its head down and climbed along the pole into the cage. The time it took for the mouse to rotate downward and climb into the cage was recorded. If the mouse fell, slipped, or failed to complete the task, the time it took to rotate downward was recorded as 30 seconds, and the time it took to climb into the cage was recorded as 60 seconds. Five consecutive trials were conducted per day, with the first two days serving as the training period and the third day serving as the test period. The time it took for the mouse in each group to rotate downward and climb into the cage during the test period was statistically analyzed.

[0157] As can be seen from the results, the time it took for the 3F-treated A53T α-synuclein transgenic mice to turn downward and climb into the cage was significantly lower than that of the PD control mice, and the 3F mice also took less time than the W20 mice (Figure 11, Panels A and B).

[0158] The novel object recognition experiment was designed based on the mouse's natural tendency to explore novel objects. The apparatus consisted of a white box measuring 40 cm × 40 cm × 40 cm. The experiment was divided into three phases: adaptation, training, and detection. In the adaptation phase, mice were placed in an empty box and allowed to spontaneously adapt for 5 min. 24 h later, the training phase began. Two identical objects were placed in the box, and the mice were allowed to habituate to the box for 5 min. Six h later, the detection phase began. The old object in the box was replaced with a new object, and each mouse was allowed to freely explore the box for 5 min. The number of times the mouse explored the new and old objects was recorded. The recognition index was calculated using the formula: (number of times in the new object - number of times in the old object) / (number of times in the new object + number of times in the old object). To avoid the influence of the mouse's odor, the box was wiped with 75% alcohol after each mouse finished exploring.

[0159] As can be seen from the results, the A53T α-synuclein transgenic mice treated with 3F showed significantly higher cognitive ability to recognize novel objects than the PD control mice, and the cognitive ability of the 3F mice to recognize novel objects was also stronger than that of the W20 mice (Figure 11C).

[0160] Example 13: 3F antibody can significantly reduce α-synuclein levels in the brains of PD transgenic mice

[0161] Phosphorylated Ser129 α-synuclein (pSer129-α-syn) is a specific pathological form of α-synuclein in the brains of PD patients. We detected pathological α-synuclein levels in the brains of A53T α-synuclein transgenic mice by immunohistochemistry using a pSer129-α-syn antibody (Abcam, catalog no. ab59264) as the primary antibody. While there was a clear pSer129-α-syn-positive area in the brainstem of PD control mice, the pSer129-α-syn-positive area in the brainstem of A53T α-synuclein transgenic mice treated with 3F was significantly reduced (Figure 12). Furthermore, 3F significantly reduced pSer129-α-syn levels in the brainstem of A53T α-synuclein transgenic mice compared with W20.

[0162] Example 14: 3F antibody can significantly increase the level of tyrosine hydroxylase in the brain of PD transgenic mice

[0163] Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis and plays an important role in dopamine signaling. There is a negative correlation between TH expression and the severity of PD. We detected TH levels in the brains of A53T α-synuclein transgenic mice using immunohistochemistry. The results showed that TH expression levels in the brainstem of A53T α-synuclein transgenic mice were significantly reduced compared with WT mice, whereas TH expression in the brainstem of PD mice was significantly elevated after treatment with 3F antibody (Figure 13). 3F significantly increased TH levels in the brains of PD transgenic mice compared with W20.

[0164] Example 15: 3F antibody significantly improves spontaneous activity and anxiety behavior in HD transgenic mice

[0165] Five-week-old male R6 / 2 mice (purchased from JACKSON LAB) were randomly divided into three groups of eight mice each. 3F, W20, or PBS (PD control) was administered intranasally once daily for a total of 28 days. Mice in the wild-type control group were administered the same amount of PBS intranasally. The mice's motor coordination ability was assessed using an open field experiment.

[0166] The open field experiment is a method for assessing spontaneous behavior, exploratory behavior, and tension in experimental animals in a novel environment. The open field was a white, open-ended plastic box (27 cm x 27 cm x 20.3 cm) divided equally into nine areas in a nine-palace grid format. A camera was attached above the box to record the mouse's movement trajectory. Mice were placed sequentially into the open field and allowed to adapt for 1 minute. After that, the mouse's movement trajectory was recorded and filmed for 30 minutes. Before each experiment, the inner walls of the box were cleaned with 70% ethanol. Three indicators were quantitatively analyzed: total path distance, number of rearings in all areas, and time spent in the central area.

[0167] The experimental results showed that the HD control mice showed a significant decrease in spontaneous activity and significant symptoms of anxiety compared to WT mice, including a decrease in the total distance traveled in the open field, the number of rearings of the hind limbs, and the time spent in the central area. Treatment with the 3F antibody significantly improved the total distance traveled, the number of rearings, and the time spent in the central area of the R6 / 2 mice (Figure 14). Furthermore, 3F significantly improved the spontaneous activity and reduced the anxiety state of the R6 / 2 mice compared to W20.

[0168] Example 16: 3F antibody can significantly reduce the level of mHTT aggregates in the brains of HD transgenic mice

[0169] Massive aggregation and deposition of mHTT protein in the nuclei and cytoplasm of neurons is an important pathological feature in the brains of HD patients or HD transgenic animals. The EM48 antibody (Merck Millipore, catalog no. MAB5374) can specifically recognize mHTT aggregates in pathological conditions. Immunofluorescence staining of R6 / 2 mouse brain tissue using the EM48 antibody revealed that there was clear EM48-positive staining in both the striatum and cortex of the brains of HD control mice. After treatment with 3F, the EM48-positive area in the brains of R6 / 2 mice was significantly reduced (Figure 15), indicating that 3F treatment effectively reduced mHTT levels in the brains of HD transgenic mice, with a stronger effect than W20.

[0170] Example 17: 3F antibody can significantly improve motor function in ALS transgenic mice

[0171] Eleven-week-old male SOD1-G93A transgenic mice (purchased from the Animal Model Institute, Nanjing University) were randomly divided into three groups of eight mice each. 3F, W20, or PBS (ALS control) was administered intranasally once daily for a total of 28 days. Mice in the wild-type control group received the same amount of PBS intranasally. The mice's motor coordination was assessed using suspension and rotarod tests.

[0172] Suspension experiment: A mouse was placed on the cage lid, and the cage lid was inverted and placed 50 cm away from the cage. The mouse was observed for 60 seconds, and the time the mouse spent hanging on the cage lid was recorded. If the mouse's stay time exceeded 60 seconds, it was recorded as 60 seconds.

[0173] The results show that the SOD1-G93A mice treated with 3F showed significantly improved suspension times compared with the ALS control mice, demonstrating that 3F can effectively enhance muscle strength in mice (Figure 16A). Furthermore, the effect of 3F was superior to that of W20.

[0174] Rotarod test: The rotarod test was conducted for three consecutive days, consisting of two phases each day: a training phase and a test phase. Training phase: The rotarod rotation speed was set at 4 rpm, and the mice were trained on the rotarod for 5 minutes, then returned to their cages and entered the test phase one hour later. Test phase: The rotarod rotation speed was set at 4 rpm within 5 minutes, and the mice were uniformly accelerated from rest to 40 rpm. The duration of the mice's time on the rotarod was recorded, and the time they did not fall was recorded as 300 seconds. Tests were conducted three times a day, with 30-minute intervals between each test. Mice from each group underwent a total of nine tests over three days, and statistical analysis was performed. The duration of the time on the rotarod for each test was analyzed statistically.

[0175] The results showed that the ALS control mice had significantly reduced rotarod duration compared with the WT mice, whereas the 3F-treated SOD1-G93A mice had significantly improved rotarod duration, and the 3F mice also had a longer rotarod duration compared with the W20 mice (Figure 16B).

[0176] Example 18: 3F antibody can significantly reduce the level of SOD1 aggregates in the brainstem of ALS transgenic mice

[0177] The brainstem of ALS patients and transgenic mice contained large amounts of mutant SOD1 aggregates. We performed SOD1 immunostaining on brain sections from SOD1-G93A mice. The results showed that SOD1 aggregates in the brainstem of 3F-treated SOD1-G93A mice were significantly reduced compared with those in the ALS control group (Figure 17). Furthermore, 3F was able to more effectively reduce the level of SOD1 aggregates in the brainstem of ALS transgenic mice than W20.

[0178] Example 19: 3F antibody significantly reduces the activation of glial cells in the brains of ALS transgenic mice

[0179] We evaluated the activation status of microglia and astrocytes in the brains of SOD1-G93A mice by immunostaining for Iba-1 and GFAP. The results showed that the activated glial cells in the brains of ALS control mice were significantly increased compared to WT mice, but 3F treatment significantly reduced the activated microglia and astrocytes in the brains of SOD1-G93A mice. 3F had a better ability to reduce glial cell activation in the brains of ALS transgenic mice than W20 (Figure 18).

[0180] Example 20: Improved antibodies such as 3F can significantly improve cognitive function in AD transgenic mice

[0181] Six-month-old male APP / PS1 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into eight groups of eight mice each and administered 3F, W20, 3A, 6A, 8H, 11G, 13A, or PBS (AD control) intranasally once daily for a total of 28 days. Mice in the wild-type control group received the same amount of PBS intranasally. The cognitive abilities of the mice were assessed using the Y-maze method described by Yu et al. (Br J Pharmacol. 2020;177:2860-2871). As can be seen from the results, mice in the AD control group showed no clear preference for the novel arm. In contrast, APP / PS1 mice in each antibody group showed a significant increase in the time spent in the novel arm (Figure 19). The 3F antibody had the most significant ability to improve cognition in AD transgenic mice.

[0182] Example 21 Specific binding of 3FI4 antibody (IgG4 subtype of 3F antibody) to Aβ oligomers

[0183] In the present invention, the 3F antibody was modified to an IgG4 subtype antibody using conventional methods and designated 3FI4 (3FI4-1, 3FI4-2, 3FI4-3, and 3FI4-4, whose light chain sequences are shown in SEQ ID No. 17, heavy chain sequences in SEQ ID Nos. 18-21, respectively, their light chain constant region sequences in SEQ ID No. 22, and heavy chain constant region sequences in SEQ ID Nos. 23-26, respectively). The binding ability of the antibody to Aβ oligomers was measured using ELISA. Specifically, Aβ oligomers were coated overnight at 4°C at 0.2 μg / well. The next day, various diluted 3FI4 antibodies were added to the coated ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and the remaining PBST was patted dry. A 1:10,000 dilution of goat anti-human HRP secondary antibody was added at 100 μL / well and incubated at 37°C for 1 h. The plate was then washed three times with PBST to remove any remaining PBST. 100 μL / well of TMB color development solution was added and developed at 37°C or room temperature. The reaction was stopped with 2 M HCl, and the absorbance at OD 450 nm was measured using a plate reader (Figure 21). The results demonstrated that the 3FI4 antibody specifically binds to Aβ oligomers.

[0184] Example 22: Aβo*3F, an Aβ oligomer that specifically binds to 3F, is extremely toxic

[0185] 22.1 Obtaining Aβ oligomers that specifically bind to 3F

[0186] 22.1.1 In Vitro Production of Aβo*3F

[0187] One mg of Aβ42, Aβ40, or other forms of Aβ (Naka Peptide Biochemicals, Inc.) was dissolved in 1 mL of 100% hexafluoroisopropanol (HFIP), vortexed for 5 min, sonicated in a water bath for 10 min, dispensed into EP tubes, allowed to evaporate overnight, and stored at -20°C. Before use, the HFIP-treated Aβ aliquot was dissolved in 50 mM NaOH at a concentration of 1 mg / mL, vortexed for 3–5 min, sonicated for 1 min, and then diluted to 10 μM with pre-chilled PBS. The solution was centrifuged at 21,000 xg for 30–40 min at 4°C, and the precipitate (approximately 5% of the initial volume) was discarded to obtain Aβ monomers. The Aβ monomers were incubated at 25°C for 2 days under static conditions and then incubated overnight at 4°C with protein A magnetic beads cross-linked with 3F antibody. On the second day, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20-100 mM glycine (pH 2.0) for 3-5 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain sAβo*3F (in vitro produced Aβos).

[0188] 22.1.2 Isolation of Aβo*3F in Brain Homogenates of APP / PS1 Mice and CSF of AD Patients

[0189] To isolate and prepare Aβos specifically recognized by 3F (Aβo*3F), brain homogenates from APP / PS1 mice and CSF samples from AD patients (derived from the First Affiliated Hospital of Zhengzhou University, who signed informed consent and received approval from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University) were incubated overnight at 4°C with protein A magnetic beads cross-linked with 3F antibody. On day 2, the magnetic beads were washed three times with 0.1% PBST and then eluted with 20 mM to 100 mM glycine (specifically, 20 mM, pH 2.0) for 3 min. This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβo*3F from 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 mixtures of Aβ aggregates obtained after 3F immunodepletion were designated Aβ-ID. sAβ-ID (produced in vitro), mAβ-ID (produced by isolation from the brains of APP / PS1 mice), and hAβ-ID (produced by isolation from the CSF of AD patients) were used as controls.

[0190] Aβ*6E10, a mixture of Aβ aggregates, was prepared by mixing brain homogenates from APP / PS1 mice or CSF from AD patients with protein G magnetic beads cross-linked with 6E10 antibodies and incubating overnight at 4°C. The mixture was then eluted with 20 mM to 100 mM glycine (specifically, 20 mM, pH 2.0) for 3 to 5 min (specifically, 3 min). This elution was repeated twice, and the eluate was neutralized to pH 7 with 1 M Tris to obtain Aβ*6E10 from APP / PS1 mouse brains (mAβ*6E10, Aβ*6E10 isolated from AD mouse brains) or Aβ*6E10 extracted from AD patient CSF (hAβ*6E10, Aβ*6E10 isolated from AD patient CSF) (Figure 22). The concentration of Aβ obtained by immunoprecipitation was measured using an Aβ detection kit.

[0191] 22.2 Characterization of the molecular weight and morphology of Aβo*3F

[0192] 500 μL of APP / PS1 mouse brain Aβo*3F (mAβo*3F) or 100 μL of SEC marker was loaded onto a Superdex 200 10 / 300 GL molecular sieve column connected to an AKTA pure system. The column was pre-equilibrated with Tris-Gly buffer and eluted at a flow rate of 0.5 mL / min. Comparison with the marker revealed that the molecular weight of mAβo*3F was 588 kDa (Figure 23A). Additionally, 10 μL of mAβo*3F was dropped onto a 200-mesh copper mesh and allowed to adsorb for 20 min, then dried by suction through a filter paper. The sample was negatively stained with 2% uranyl acetate for 30 s, dried by suction through a filter paper, and air-dried. The sample was examined under a transmission electron microscope (TEM, Hitachi H7700, Japan) at a 120 kV operating voltage and 100,000× magnification. As can be seen from the results, mAβo*3F was a particle with a diameter of approximately 10 nm (Figure 23A). The molecular weight and distribution of sAβo*3F and Aβo*3F extracted from CSF of AD patients (hAβo*3F) were consistent with those of mAβo*3F.

[0193] 22.3 Strong cytotoxicity of Aβo*3F

[0194] As can be seen from the MTT results, the 50% inhibitory concentration (IC50) of sAβo*3F toxicity against N2a cells was approximately 0.186 nM, whereas the IC50 of sAβos before immunoprecipitation was approximately 63.26 nM, a 340-fold difference between the two (Figures 23B and 23C). The IC50 of mAβo*3F against N2a cells was approximately 0.111 nM, as detected by the MTT method, and the IC50 of hAβo*3F against primary neurons was approximately 0.057 nM (Figure 23D). The IC50 of hAβo*3F against primary neurons was approximately 0.076 nM (Figure 23E). As can be seen from the results, Aβo*3F is a highly neurotoxic oligomer.

[0195] 22.3 Aβo*3F significantly impairs cognitive function in mice and severely impairs neuronal function in the mouse brain.

[0196] Three-month-old C57BL / 6 mice were divided into six groups of six to eight mice each and injected into the lateral ventricle. The groups were: 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. Behavioral and cognitive performance was monitored 24 h after injection, and the mice were then dissected for pathological analysis of the brain sections.

[0197] As can be seen from the Y-maze results, the duration in the novel arm of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 was significantly reduced compared to the control group. In contrast, there was no significant difference in the duration of the novel arm in mice injected with 2.5 nM mAβ*6E10, 2.5 nM mAβ*ID, or 600 nM mAβ*ID compared to the control group. mAβo*3F, but not mAβ*6E10 or mAβ-ID, was able to induce severe memory impairment in mice at low concentrations, while mAβ*6E10 was only able to induce similar memory impairment at concentrations up to 600 nM (Figure 24A).

[0198] Mouse brain samples were stained using the FD Rapid Golgi Staining Kit. Dendritic spine density was calculated and found to be significantly reduced in neurons in the CA1 region of the brains of mice treated with 2.5 nM mAβo*3F and 600 nM mAβ*6E10 compared with the control group, whereas 2.5 nM mAβ*6E10, 2.5 nM mAβ-ID, or 600 nM mAβ-ID had no significant effect on dendritic spine density in neurons in the brains of mice (Figures 24B and 24C).

[0199] Example 23 Further optimization and activity measurement of 3F antibody

[0200] The 100th amino acid, K, of the 3F antibody was mutated to R (if the MA residue introduced for expression as a single-chain antibody is not taken into account, it is considered to be the 98th amino acid in the antibody numbering, and this mutant is designated K98R mutant, the sequence of which is shown in SEQ ID NO: 27), and the effect of the K98R mutation on the affinity of the single-chain antibody was examined. The method of use was as follows.

[0201] The above Aβ oligomers were coated onto a 96-well plate at 100 ng / well and left overnight at 4°C. The next day, the plate was blocked with 3% BSA for 2 hours at room temperature, washed twice with PBST, and then tapped dry. The K98R mutant and 3F were diluted 14 times starting from 1 μg / mL. The diluted antibody solution was added to the ELISA plate coated with the above Aβ oligomers, and the plate was incubated in multiple wells at 37°C for 1 hour, washed three times with PBST, and tapped dry. Goat anti-human IgG-HRP diluted 1:10,000 was added, and the plate was incubated for 45 minutes at 37°C, washed three times with PBST, and tapped dry. TMB color development solution was added for approximately 15 minutes, and the color was stopped with stop solution, followed by OD analysis. 450nm The data was read out using , and the values were analyzed and compared using graphpad, and plotted.

[0202] The results are shown in Figure 25. As can be seen from the experiment, after mutating K at position 98 of the heavy chain of the single-chain antibody to R, the affinity was further improved, suggesting that the 98th amino acid plays an important role in the binding process of this single-chain antibody with Aβ oligomers. The inventors investigated the cause of this result and found that, when numbered according to IMGT, the 98th amino acid is also a residue of the heavy chain CDR3 of the 3F antibody (when numbered according to IMGT, the CDR sequences of the heavy and light chains 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 further investigated the in vivo activity of the K98R mutant and found that it was even more improved than that of the 3F antibody (data not shown).

[0203] Equivalent Scheme

[0204] While various embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of numerous other means and / or structures for achieving the functions described herein and / or obtaining one or more of the results and / or advantages described herein, and all such variations and / or modifications are intended to be included within the scope of the present invention. More broadly, those skilled in the art will readily appreciate that all parameters, materials, and settings described herein are exemplary, and that the actual parameters, materials, and / or settings will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalent schemes to the specific embodiments of the present invention described herein. Thus, it should be understood that the foregoing embodiments and examples are presented merely by way of example, and that the invention may be practiced otherwise than as specifically described and claimed, within the scope of the appended claims and their equivalents. Any combination of two or more such features, systems, articles, materials, and / or methods, if not mutually inconsistent, is within the scope of the present invention.

[0205] The term "and / or," as used in the present specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjoined in some cases but not in other cases. In addition to the elements specifically identified by the term "and / or," other elements may optionally be present, whether associated with those specifically identified elements or not, unless expressly indicated otherwise. Thus, as a non-limiting example, when used in conjunction with an open term such as "comprising," a reference to "A and / or B" can, in one embodiment, refer to A without B (optionally including elements other than B), in another embodiment, refer to B without A (optionally including elements other than A), in yet another embodiment, include both A and B (optionally including other elements), and so forth.

[0206] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or," as defined above. For example, when separating items in a list, "or" or "and / or" should be understood to be inclusive, i.e., the inclusion of at least one of a plurality of elements or a list of elements, but including more than one, including a plurality of elements and optionally other items not listed. When used in the claims, terms clearly indicating the contrary, such as "only one of" or "exactly one of," "consisting of" refers to the inclusion of exactly one element of a plurality of elements or a list of elements. Generally, as used herein, the term "or" should only be understood as indicating an exclusive alternative scheme (i.e., "one or the other, but not both") when preceded by terms of exclusion, such as "either," "one of," "only one of," or "exactly one." "Consisting essentially of" when used in the claims should have its ordinary meaning in the field of patent law.

[0207] As used herein and in the claims, when referring to a list of one or more elements, the term "at least one" should be understood to mean at least one element selected from any one or more elements of the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the term "at least one" refers, may optionally be present, whether or not related to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B" or equivalently "at least one of A and / or B") can mean, in one embodiment, at least one, optionally more than one, A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally more than one, B, and no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements); etc.

[0208] In the claims and the above specification, all conjunctions such as "comprising," "including," "with," "comprises," "containing," "relating to," "having," etc. are understood to be open, i.e., to mean including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively.

[0209] The use of sequence terms in the claims, such as "first," "second," "third," etc., to modify the claim elements themselves does not imply any priority, precedence, or order of one claim element relative to another claim element, or the chronological order in which actions are performed in a method, but is used solely as a tag (but used in numerical terms) to distinguish one claim element with a certain name from another element with the same name to distinguish the claim elements.

Claims

1. and having amino acid substitutions at one or more positions selected from the 226th, 227th, and 228th amino acid residue positions of the W20 antibody, the amino acid sequence of which is set forth in SEQ ID No. 1, and the amino acid positions of the antibody are numbered according to the Kabat numbering system. Improved amyloid protein oligomer-specific antibodies or antigen-binding fragments thereof.

2. The 226th amino acid residue of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is substituted with a non-polar and hydrophobic amino acid similar in properties to alanine, preferably with glycine, valine, leucine, isoleucine, phenylalanine, tryptophan, or proline, more preferably with glycine, phenylalanine, or tryptophan.

2. The improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to claim 1.

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

4. The 228th amino acid residue of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is substituted with an aliphatic amino acid whose structure is 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.

4. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. the amino acid residues 226 to 228 of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof are substituted with AVR, GSR, WVR, FER, NFR, or VRR, respectively; 5. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. the 224th and 225th amino acid residues of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof are substituted with glutamine and threonine, respectively; 6. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is an antigen-binding fragment, preferably an scFv, F(ab') 2 , Fab', Fab, Fd, Fv, bispecific antibodies, camelid antibodies, CDRs, and antigen-binding fragments selected from the group consisting of smallest antibody recognition units (dAb), more preferably scFv, F(ab') 2 , Fab', and Fab, 7. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, and preferably, the antibody is selected from the group consisting of IgG, IgM, IgA, IgD, IgE, and subtypes thereof, more preferably, the antibody is selected from the group consisting of IgG1-4 subtypes, and more preferably, the antibody is an IgG4 subtype.

7. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

9. The improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is an scFv, and its amino acid sequence is set forth in any one of SEQ ID Nos. 5, 4, and 6 to 9.

9. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. The sequence of the CDR3 of the light chain of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is shown in SEQ ID No.

15.

10. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. The improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof has an arginine substitution at position 98 relative to the 3F antibody, and preferably, the amino acid sequence of the heavy chain of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is set forth in any one of SEQ ID Nos. 33-36, and the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof is an scFv, and the amino acid sequence thereof is set forth in SEQ ID No.

27.

10. An improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

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

13. An expression vector comprising the nucleic acid molecules of claim 12 operatively ligated together.

14. A host cell comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13.

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

16. Cultivating the host cell of claim 14 under culture conditions suitable for expression of the improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof, and optionally isolating and purifying the resulting product. A method for producing an improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

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

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

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

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

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

22. In the manufacture of a drug for specifically binding to Aβo*3F, a highly toxic amyloid protein oligomer, in a subject and suppressing its neurotoxicity, Use of an improved amyloid protein oligomer-specific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a nucleic acid molecule according to claim 12, an expression vector according to claim 13, a host cell according to claim 14 or a composition according to claim 15, The highly toxic amyloid protein oligomer Aβo*3F was isolated from a mixture of Aβ oligomers by immunoprecipitation against 3F antibody, and its typical characteristics are that of a high-molecular-weight Aβ oligomer, with a molecular weight of approximately 588 kDa and a diameter of approximately 10 nm based on analysis by size exclusion chromatography (SEC).

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