Scfv antibody that specifically binds aβ, and use thereof

Abstract

Provided are an scFv antibody that specifically binds Aβ, an isolated nucleic acid encoding the antibody, a vector comprising the nucleic acid, a cell comprising the isolated nucleic acid or the vector, a method for preparing the scFv antibody that specifically binds Aβ, and a use of the scFv antibody that specifically binds Aβ in the preparation of a medicament for treating Alzheimer's disease.

Description

scFv antibodies that specifically bind to Aβ and their applications Technical Field

[0001] This application relates to the field of antibody technology. Specifically, it relates to scFv antibodies that specifically bind to Aβ and their uses. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by functional impairment of cognition and memory. Since its discovery by German neurologist Alois Alzheimer in 1906, AD has become a globally prevalent age-related disease with a huge disease burden. The cost of treatment for Alzheimer's disease has reached one trillion US dollars annually, and this figure is expected to double by 2030 as the prevalence increases, placing enormous economic and social pressure on individuals, families, and society.

[0003] The pathogenesis of AD is not yet clear, but the β-amyloid peptide (Aβ) cascade hypothesis and the tau protein hypothesis are widely accepted by the academic community.

[0004] Aβ is derived from the hydrolysis of β-amyloid precursor protein (APP), produced by various cells, and circulates in the blood, cerebrospinal fluid, and cerebrospinal fluid. Most of it is bound to chaperone protein molecules, with a small number existing in a free state. The most common subtypes are Aβ40 and Aβ42. Aβ42 is more toxic and more prone to aggregation, forming the core of Aβ precipitates and inducing neurotoxic effects.

[0005] Tau protein is the most abundant microtubule-associated protein. The microtubule system is a component of the neuronal cytoskeleton and participates in various cellular functions. Microtubules are composed of tubulin and microtubule-associated proteins. In the normal brain, the cellular functions of tau protein are to bind to tubulin to promote its polymerization and form microtubules; to bind to the formed microtubules, maintain microtubule stability, reduce the dissociation of tubulin molecules, and induce microtubule bundle formation.

[0006] The two most important pathological features of Alzheimer's disease (AD) are extracellular amyloid plaque deposition and neurofibrillary tangles (NFT). Neurofibrillary tangles are formed by the thickening and twisting of neurofibrils, confirmed by silver-stained electron microscopy to consist of double-helicaled filaments. They are most commonly found in larger neurons, particularly in pyramidal cells of the hippocampus, amygdala, medial temporal lobe, and frontal cortex. This change is a marker of neuronal death. Excessive Aβ deposition in the brain leads to hyperphosphorylation of tau protein, neurofibrillary tangles, loss of synaptic cell function, neuronal death, and cognitive impairment.

[0007] Currently, drugs for Alzheimer's disease are mainly divided into three categories: acetylcholinesterase inhibitors, NMDA receptor antagonists, and anti-Aβ monoclonal antibodies. Acetylcholinesterase inhibitors and NMDA receptor antagonists can only alleviate symptoms and slow disease progression, but cannot directly treat the disease, and they have significant side effects including digestive, nervous, and cardiovascular complications. Therapies targeting soluble Aβ include gamma-secretase inhibitors, BACE1 inhibitors, and anti-Aβ monoclonal antibodies. While gamma-secretase inhibitors and BACE1 inhibitors, which have entered phase 3 clinical trials, can reduce Aβ production to some extent, they have not significantly affected amyloid protein deposition in the brain. Moreover, because gamma-secretase and BACE1 also mediate the cleavage of other proteins, these inhibitors have shown toxic side effects in clinical trials. Therefore, anti-Aβ monoclonal antibodies are currently the most promising clinical treatment for Alzheimer's disease.

[0008] Current challenges in the development and phase III clinical trials of anti-Aβ monoclonal antibodies, such as excessively strong accompanying neuroimmunoinflammatory reactions, vasogenic cerebral edema, and cerebral hemorrhage, limit their use. Meanwhile, peripheral antibody molecules enter the brain parenchyma via transcytosis through receptor-ligand binding at the blood-brain barrier. This is an energy-intensive active transport process, and due to the large molecular weight of IgG (>150kDa), this pathway is not very active. Pharmacokinetic data from aducanumab (trade name Aduhelm) and its competitors show that the brain penetration efficiency of conventional IgG-mAbs is less than 1%.

[0009] Single-chain variable fragments (scFvs) consist of a heavy chain variable region (VH) and a light chain variable region (VL) linked by a linker peptide. Each VH and VL in the scFv structure contains four framework regions (FRs) and three complementarity-determining regions (CDRs). The CDRs are crucial for antibody recognition and antigen binding, determining antibody specificity. In scFvs, VH and VL are typically linked by a linker peptide of 15 to 25 amino acids, rich in hydrophilic amino acids such as glycine and serine, forming a (GGGGS)n-like structure, thus providing sufficient flexibility for the peptide chain. Although scFvs lack the constant regions of antibodies, they can still stably bind to specific antigens and function. Compared to full-length antibodies, scFvs exhibit superior permeability across endothelial and epithelial barriers and, due to their smaller molecular weight, can diffuse more rapidly through the tissue matrix. Furthermore, the low immunogenicity, short in vivo retention time, and ease of expression of scFv make it an ideal targeting molecule, widely used in disease diagnosis and treatment. scFv can fuse with fluorescent proteins for disease diagnosis; it can also bind with chemical toxins to form immunotoxins for cancer treatment.

[0010] In view of this, there is an urgent need in the art for an anti-Aβ monoclonal scFv antibody that combines the promise of anti-Aβ monoclonal antibodies in the treatment of Alzheimer's disease with the advantages of scFv antibodies. Summary of the Invention

[0011] In order to invent new anti-Aβ monoclonal antibodies and improve their blood-brain barrier permeability and reduce immunogenicity, the inventors designed and developed a novel monoclonal scFv antibody (scFv-ANG) targeting the Aβ oligomeric antigenic epitope. This antibody can efficiently bind to the LRP1 receptor in the blood-brain barrier and be transported into the brain. It binds to Aβ monomers and oligomers in the brain, blocking the dimerization of Aβ monomers and the aggregation of Aβ oligomers, thereby promoting the depolymerization and clearance of Aβ plaques.

[0012] scFv antibodies that specifically bind to Aβ

[0013] In a first aspect, this application provides an scFv antibody that specifically binds to Aβ, comprising a heavy chain variable region (VH), a linker peptide, a light chain variable region (VL), and a 3' terminal sequence, wherein:

[0014] The heavy chain variable region includes the following complementary determinant region (CDR):

[0015] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1.

[0016] The amino acid sequence is as shown in SEQ ID NO: 2, HCDR2, and

[0017] The amino acid sequence is HCDR3 as shown in SEQ ID NO: 3;

[0018] The light chain variable region includes the following complementary determinant region (CDR):

[0019] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4.

[0020] The amino acid sequence is LCDR2 as shown in SEQ ID NO: 5, and

[0021] The amino acid sequence is LCDR3 as shown in SEQ ID NO: 6;

[0022] The amino acid sequence of the linker peptide is shown in SEQ ID NO: 9; and

[0023] The amino acid sequence of the 3' terminal sequence is shown in SEQ ID NO: 10.

[0024] HCDR1 amino acid sequence (SEQ ID NO: 1): SGFAFSSY

[0025] HCDR2 amino acid sequence (SEQ ID NO: 2): LEWVAVIWFDGTKKYYT

[0026] HCDR3 amino acid sequence (SEQ ID NO: 3): DRGIGARRGPYYMD

[0027] LCDR1 amino acid sequence (SEQ ID NO: 4): QSISS

[0028] LCDR2 amino acid sequence (SEQ ID NO: 5): PKLLIYAASSLQ

[0029] LCDR3 amino acid sequence (SEQ ID NO: 6): SYSTPL

[0030] Linker peptide amino acid sequence (SEQ ID NO: 9): GSTGSGSGKPGSGEGSTKG

[0031] The amino acid sequence of the 3' terminal sequence (SEQ ID NO: 10):

[0032] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 7; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8.

[0033] In a further embodiment, the full-length amino acid sequence of the scFv antibody that specifically binds to Aβ is shown in SEQ ID NO: 11. Heavy chain variable region amino acid sequence (SEQ ID NO: 7):

[0034] Light chain variable region amino acid sequence (SEQ ID NO: 8):

[0035] The full-length amino acid sequence of the scFv antibody (SEQ ID NO: 11):

[0036] Pharmaceutical Composition

[0037] The scFv antibody that specifically binds to Aβ according to this application can be formulated into a pharmaceutical composition for administration to a subject (e.g., for prevention or treatment of a disease according to this application). Therefore, this application provides a pharmaceutical composition comprising:

[0038] Effective amounts of the scFv antibody that specifically binds to Aβ as described in any of the preceding claims for prevention or treatment, and

[0039] One or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0040] Typically, pharmaceutical compositions include pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0041] In some embodiments, "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein along with other components, such as physiological / pharmaceutical-grade carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0042] In some implementations, the antibody described herein is an scFv antibody that specifically binds to Aβ.

[0043] Drug formulation is a well-known technique, as further described, for example, in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins (2000); and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippincott, Williams & Wilkins Publishers (1999).

[0044] Pharmaceutical compositions may also be in various forms, including, for example, liquid, semi-solid, and solid dosage forms. Examples include liquid solutions (e.g., injectable and infusionable solutions), suspensions, tablets, pills, powders, liposomes, and suppositories. Preferred forms may depend on the administration method and therapeutic application. Typically, the pharmaceutical compositions described herein are in the form of injectable or infusionable solutions.

[0045] In one embodiment, the scFv antibody that specifically binds to Aβ according to this application is provided in a buffer solution at an appropriate concentration and stored at 2°C–8°C.

[0046] The pharmaceutical composition may be administered via parenteral route (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, "parenteral administration" includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some specific embodiments, intravenous administration of scFv antibodies that specifically bind to Aβ is performed.

[0047] In some implementations, scFv antibodies that specifically bind to Aβ can be prepared together with controlled-release carriers (such as controlled-release formulations, implants, or microencapsulated delivery systems). Methods for preparing such formulations are generally known.

[0048] isolated nucleic acids

[0049] This application also provides isolated nucleic acids encoding scFv antibodies that specifically bind to Aβ as described in any of the preceding claims.

[0050] In some embodiments, the sequence of the isolated nucleic acid is as shown in SEQ ID NO: 12, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 12.

[0051] It should be understood that even though this application provides specific examples of nucleotide sequences, the nucleotide sequences that can "encode the scFv antibody that specifically binds to Aβ of this application" are not limited to these specific sequences, because the same amino acid sequence can be encoded by equivalent nucleotide sequences depending on the codon preference and codon degeneracy of the expressing host.

[0052] The nucleic acid sequence encoding an scFv antibody that specifically binds to Aβ (SEQ ID NO: 12)

[0053] expression carrier

[0054] This application also provides an expression vector comprising the nucleic acid isolated according to this application.

[0055] Nucleic acids encoding target antibodies or their fragments can be constructed, introduced into expression vectors, and expressed in suitable host cells.

[0056] host cells

[0057] This application also provides a host cell containing the aforementioned nucleic acid or expression vector for expressing the scFv antibody that specifically binds to Aβ as described in this application.

[0058] Host cells include prokaryotic and eukaryotic host cells. Eukaryotic host cells include, but are not limited to, mammalian cells, insect cells, plant cells, and fungal cells. Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Exemplary host cells include, but are not limited to, CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocellular carcinoma cells, A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast, such as, but not limited to, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, and *Kluyveromyces*.

[0059] In this application, the host cell cannot develop into a complete animal or plant individual.

[0060] Methods of generating antibodies

[0061] Antibodies can be produced in bacteria or eukaryotic cells. Antibodies can be produced in bacterial cells or eukaryotic cells (e.g., CHO, 293E). To produce the target antibody, a nucleic acid encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover the antibody.

[0062] If antibodies are expressed in animal cells (such as CHO cells), the expression vector includes the required promoter, such as the SV40 promoter, EF1α promoter, or CMV promoter. In addition to the nucleic acid sequence encoding the antibody or its domains, recombinant expression vectors can carry additional sequences, such as selection markers. Selection markers facilitate the selection of host cells to which the vector has been introduced. For example, the marker is often used to select resistance to drugs such as G418, hygromycin, or methotrexate.

[0063] In some implementations, antibodies are generated in mammalian cells. Indicative mammalian host cells used for antibody expression include Chinese hamster ovary (CHO cells), human embryonic kidney 293 cells, COS cells, NIH3T3 cells, NSO myeloma cells, and SP2 cells.

[0064] The antibody of this application can be isolated from host cells and purified to substantially pure antibody. Separation and purification methods commonly used for antibody purification can be applied to the separation and purification of antibodies, such as chromatography, filtration, ultrafiltration, salting out, precipitation, extraction, distillation, electrophoresis, isoelectric point focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity, ion exchange, hydrophobic, reversed-phase, and adsorption chromatography. Columns used for affinity chromatography include, for example, protein A columns and protein G columns.

[0065] In some implementations, antibodies are produced by chemical synthesis, which includes steps such as synthesizing the amino acid sequence of the antibody.

[0066] Prevention or treatment methods

[0067] The scFv antibody that specifically binds to Aβ in this application may be used to treat or prevent Alzheimer's disease.

[0068] In some implementations, Alzheimer's disease is familial or early-onset Alzheimer's disease.

[0069] In some implementations, the Alzheimer's disease is associated with abnormal expression, abnormal activity, and abnormal deposition of Aβ.

[0070] This application provides a method for preventing or treating a disease, the method comprising administering to a subject a preventive or therapeutically effective amount of a scFv antibody, nucleic acid, or pharmaceutical composition that specifically binds to Aβ.

[0071] In some implementations, the disease is Alzheimer's disease.

[0072] This application also provides a scFv antibody, nucleic acid, or pharmaceutical composition that specifically binds to Aβ for the prevention or treatment of disease.

[0073] In some implementations, the disease is Alzheimer's disease.

[0074] Administering a specific Aβ-binding scFv antibody for a specified period of time to subjects at risk of Alzheimer's disease, diagnosed with, or with Alzheimer's disease provides a therapeutic effect. The specific Aβ-binding scFv antibody can be administered alone (monotherapy) or in combination with a second therapeutic agent (combination therapy).

[0075] In the specific implementation plan, the subjects are individuals who are susceptible to, suspected of having, or already have the disease.

[0076] "Treatment" refers to providing a subject with an scFv antibody that specifically binds to Aβ, or a pharmaceutical composition thereof. The subject has symptoms of one or more diseases. Typically, the scFv antibody that specifically binds to Aβ is administered to the treated subject or a group of subjects in an amount that effectively relieves disease symptoms.

[0077] In specific implementation schemes, those skilled in the art can determine the selection of an effective dose based on consideration of a variety of factors (e.g., through clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the subject's weight, the subject's immune status, and other factors known to those skilled in the art.

[0078] The effective dosage in a specific implementation can be obtained from dose-response curves derived from animal model testing systems, and can be determined based on the physician's judgment and the individual subject's condition. As an example, the amount of drug required for a single administration to a subject can be conveniently obtained by calculating the product of the subject's weight and the dose per unit body weight required for that single administration. For instance, in drug preparation, an adult's weight is generally considered to be 50-70 kg, and the dosage can initially be determined using the equivalent dose conversion relationship between experimental animals and human body weight doses. Alternatively, it can be determined according to guidelines issued by drug regulatory agencies such as the SFDA and FDA. In some implementations, the dosage for humans and mice can be converted using a conversion factor of 0.0026 based on the body surface area of ​​humans and mice.

[0079] The active ingredient described herein (e.g., an scFv antibody that specifically binds to Aβ) may be administered in a single dose or divided into many smaller unit doses administered at regular time intervals. It should be understood that the dosage, duration, and intervals of treatment are functions of the disease being treated and can be inferred from animal or clinical trial data. Administration may include a single dose or two or more doses at appropriate time intervals. Intervals between consecutive doses may be 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0080] use

[0081] On the other hand, this application also provides the use of scFv antibodies, nucleic acids or pharmaceutical compositions that specifically bind to Aβ in the preparation of medicaments for treating or preventing diseases.

[0082] In some implementations, the disease is Alzheimer's disease.

[0083] This application also provides scFv antibodies, nucleic acids, or pharmaceutical compositions that specifically bind to Aβ for use as medicaments. In some embodiments, this application also provides scFv antibodies, nucleic acids, or pharmaceutical compositions that specifically bind to Aβ for use as medicaments for treating diseases.

[0084] In some embodiments, the aforementioned scFv antibody that specifically binds to Aβ is provided for treating the disease, wherein the scFv antibody that specifically binds to Aβ is administered in combination with a second therapeutic agent.

[0085] In some implementations, the scFv antibody that specifically binds to Aβ and the second therapeutic agent are in the same or different containers.

[0086] In some embodiments, the use of the scFv antibody that specifically binds to Aβ, or the pharmaceutical composition described in this application, in the preparation of a medicament for reducing Aβ plaque burden, plaque number, and plaque diameter.

[0087] In some embodiments, the use of the scFv antibody that specifically binds to Aβ as described in this application, or the pharmaceutical composition described in this application, in the preparation of a medicament for improving spatial memory and cognitive function.

[0088] In some embodiments, the scFv antibody that specifically binds to Aβ described in this application, or the pharmaceutical composition described in this application, can be used as a drug to reduce Aβ plaque burden, plaque number, and plaque diameter, or to improve spatial memory and cognitive function.

[0089] Detection methods

[0090] This application provides a method for determining Aβ levels in a sample in vitro, including the following steps:

[0091] This allows the sample to come into contact with an effective amount of scFv antibody that specifically binds to Aβ.

[0092] In some specific embodiments, the Aβ-specific scFv antibody described in this application is used for immunoassays. Immunoassays involve detecting the level of Aβ in a biological sample from a subject by contacting the Aβ-specific scFv antibody of this application with the biological sample. Typically, the Aβ-specific scFv antibody of this application may be conjugated to a fluorescent tag or other tag.

[0093] In this application, the "method for determining Aβ levels in a sample in vitro" does not involve the diagnosis of a disease.

[0094] reagent kit / pharmacy kit

[0095] This application provides a kit comprising an scFv antibody that specifically binds to Aβ, a pharmaceutical composition, a nucleic acid, an expression vector, or a host cell; the kit is used for diagnosing or detecting Aβ protein in an in vitro sample or cells expressing Aβ protein.

[0096] This application also provides a kit comprising at least one container, each container independently containing: an scFv antibody that specifically binds to Aβ, a nucleic acid, or a pharmaceutical composition.

[0097] For diagnostic or research use, the kit contains one or more of the following components: analytical reagents, buffer solutions, and the antibody described in this application. Additionally, these reagents / kits may include instructions for use.

[0098] This application modifies the anti-Aβ monoclonal antibody by scFv modification, which improves the antibody's antigen recognition ability. The scFv antibody that specifically binds to Aβ in this application can improve spatial memory and cognitive function in vivo, reduce the Aβ plaque burden, number of plaques and plaque diameter in the hippocampus and cortex, and effectively reduce neuroinflammatory response, which is a novel treatment for Alzheimer's disease. Attached Figure Description

[0099] Figure 1 shows the SDS-PAGE analysis results of the prepared and purified antibody.

[0100] Figure 2 shows the affinity verification results of the prepared and purified antibody.

[0101] Figure 3 shows the administration method and timeline. 5XFAD mice were divided into 3 groups of 10 mice each. Starting from week 28, each group was injected weekly via tail vein with the specific Aβ-binding scFv antibody (scFv-ANG, concentration 2 mg / ml, dose 10 mg / kg), PBS, or aducunabumab (concentration 2 mg / ml, dose 10 mg / kg). After 4 weeks of administration, behavioral experiments, including new object recognition, Y maze, and Morris water maze, were conducted.

[0102] Figures 4A to 4D show the results of the Y-maze experiment. The Y-maze test assessed the improvement in spatial memory and cognitive function in 5XFAD mice treated with scFv antibody. Figure 4A shows the experimental pattern of the Y-maze, Figure 4B shows that there was no difference in the total number of arm entries, Figure 4C shows the changes in the spontaneous alternation rate among different groups, and Figure 4D shows the Y-maze trajectory diagrams of different drug administration groups.

[0103] Figures 5A to 5D show the results of the novel object recognition experiment. The novel object recognition experiment examined the improvement in spatial memory and cognitive function in 5XFAD mice after treatment with scFv antibodies. Figure 5A shows the training and trial patterns for novel object recognition, Figure 5B shows the total number of explorations, Figure 5C shows the differences in cognitive indices between different groups, and Figure 5D is a trajectory diagram of the objects explored by the 5XFAD mice.

[0104] Figures 6A to 6E show the results of the Morris water maze experiment. The Morris water maze experiment assessed the improvement in spatial memory and cognitive function in 5XFAD mice after treatment with scFv. Figure 6A is a schematic diagram of the Morris water maze, Figure 6B shows the trajectory diagrams between different drug administration groups in Test 1 and Test 2, Figure 6C shows the changes in swimming speed of mice at different training days, Figure 6D shows the time it took for 5XFAD mice in different drug administration groups to reach the platform, and Figure 6E shows the number of times the platform was crossed after its removal.

[0105] Figures 7A to 7F show the results of multicolor immunofluorescence. Immunofluorescence staining detected a reduction in Aβ plaque burden in 5XFAD mice after treatment with scFv antibody. Figure 7A shows the immunofluorescence staining of Aβ in the hippocampus and cortical regions; Figures 7B to 7E show statistical graphs of plaque burden, number of plaques, and plaque diameter; Figure 7F shows the Aβ plaque staining map in the sagittal plane of the whole brain and a magnified view of the hippocampus. Detailed Implementation

[0106] the term

[0107] The terminology used herein is for descriptive purposes only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0108] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of,” for example, for VH comprising HCDR1 as shown in SEQ ID NO: 1, which explicitly covers the amino acid sequence HCDR1 as shown in SEQ ID NO: 1.

[0109] It should be understood that the ordinal numbers “first”, “second”, “Formula 1”, “Formula 2”, “1)”, “2)”, etc. in this application are only used to distinguish different technical features, elements, components and steps, and are not intended to impose restrictions on level, order or quantity.

[0110] The target refers to the object targeted by the therapeutically active ingredient (the scFv antibody that specifically binds to Aβ) of this application. The target can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isotype, modified form, free, or surface-expressed). In this application, the target specifically refers to a protein. As an example, the scFv antibody that specifically binds to Aβ targets Aβ.

[0111] The nucleotide or amino acid information of Aβ, which is the target, is well known in the art and can be obtained, for example, but not limited to, from literature or databases.

[0112] In the context of this application, Aβ should be interpreted broadly to include all forms of Aβ molecules at various stages, such as, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the Aβ gene, including cDNA, mRNA, precursor proteins, mature proteins, and fragments thereof. In specific embodiments, Aβ refers to human Aβ. In some specific embodiments, human Aβ refers to mature Aβ protein, particularly Aβ oligomers.

[0113] In the field of antibodies, targets typically exist in the form of antigens. An "antigen" is a molecule or part thereof that can be selectively recognized or bound by antigen-binding molecules (such as scFv antibodies that specifically bind to Aβ). An antigen may have one or more epitopes.

[0114] An epitope is a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformational epitopes). Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids. Antibodies or their antigen-binding fragments that bind to specific epitopes can be screened using methods routine in the art, such as, but not limited to, alanine scanning, peptide cleavage analysis, epitope extraction, and chemical modification of the antigen.

[0115] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and full-length antibodies, as long as they exhibit the desired antigen-binding activity. Typically, natural IgG antibodies are heterotetrameric proteins composed of two light chains and two heavy chains linked by disulfide bonds. From the N to C terminus, each heavy chain has one variable region (VH) and three constant domains (CH1, CH2, and CH3). From the N to C terminus, each light chain has one variable region (VL) and one constant light domain (CL). The specific meaning of "antibody" can be determined by a person skilled in the art based on the context.

[0116] "Specific binding" means that, compared to other antigens or epitopes, the scFv antibody of this application that specifically binds to Aβ binds to the targeted antigen or epitope with a higher affinity. Typically, approximately 1 × 10⁻⁶. -7 M or a smaller equilibrium dissociation constant (K) D K is considered a "specific" binding. Known methods can be used to measure K. D For example, FACS or surface plasmon resonance. "Specific binding" does not exclude cross-reactivity with homologous antigens of other species (such as cynomolgus monkeys (Macaca fascicularis), chimpanzees (Pan troglodytes), or marmosets (Callithrix jacchus). Therefore, "specific binding to human Aβ" or "specific binding to Aβ" means that the scFv antibody that specifically binds to Aβ can specifically recognize or bind to Aβ (or its epitopes, especially Aβ oligomeric antigenic epitopes).

[0117] "Affinity" is used to describe the strength of the non-covalent interaction between an scFv antibody that specifically binds to Aβ and its antigen or epitope. Affinity is typically expressed by the equilibrium dissociation constant (K0). D The symbols ) represent the association rate of a specific antibody-antigen interaction. "kassoc" or "ka" refers to the association rate of that interaction, while "kdis" or "kd" refers to the dissociation rate of that interaction. "K" indicates the rate of dissociation. D The ratio of kd to ka (i.e., kd / ka) is expressed as molar concentration.

[0118] An "antibody fragment" or "antigen-binding fragment" is different from a complete antibody molecule; it contains a portion of the complete antibody that retains the antigen-binding ability of the complete antibody.

[0119] A single-chain variable fragment (scFv) is composed of a heavy chain variable region (VH) and a light chain variable region (VL) linked by a linker peptide.

[0120] "Variable region" refers to the domain in an antibody or antigen-binding fragment that binds to the antigen.

[0121] The "complementarity-determining region" or "CDR" refers to the region within the variable region that primarily facilitates antigen binding. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. From the N-terminus to the C-terminus, each VH and VL region is named as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0122] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the Kabat numbering system (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering system, and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278).

[0123] The correspondences between various numbering systems are well known to those skilled in the art. In other words, when a CDR sequence and its location in an antibody are provided under one numbering system, those skilled in the art are able to determine the corresponding CDR sequence and its location in an antibody under another numbering system. Technical solutions corresponding to different numbering systems are considered equivalent technical solutions.

[0124] Unless otherwise stated, in this application, references to the positions of residues in the antibody variable region and CDR refer to the positions numbered according to the Kabat numbering system.

[0125] In view of the foregoing, those skilled in the art will understand that when describing the scFv antibody that specifically binds to Aβ according to the Kabat numbering system, it also includes the sequence corresponding to the Chothia, AbM CDR, or IMGT numbering system.

[0126] The term "percentage of amino acid sequence identity (%)" or simply "identity" is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage of amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0127] As an example, "the amino acid sequence of the heavy chain variable region has at least 85% sequence identity with SEQ ID NO: 7" means that the amino acid sequence includes CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3, and that amino acid mutations are allowed to be introduced in regions outside the CDRs, so that the amino acid sequence of the heavy chain variable region has at least 85% sequence identity with SEQ ID NO: 7.

[0128] "Amino acid mutation" includes amino acid substitution, deletion, insertion, modification, or any combination thereof. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided the final construct possesses the desired properties. Amino acid substitutions can be introduced into antibodies of interest, and the product can be screened for desired activities, such as retention / improvement of antigen binding or reduced immunogenicity. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. In one embodiment, an amino acid mutation is a non-conserved amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc.

[0129] The term "nucleic acid" is used interchangeably with "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The nucleic acids are synthetic, naturally occurring, and non-natural. Unless otherwise specified, nucleic acid sequence also encompasses its conserved variants (e.g., degenerate substitutions) and complementary sequences, as well as explicitly stated sequences. The term "nucleic acid molecule" includes multiple nucleic acid molecules.

[0130] A "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide it is linked to. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which a foreign DNA segment is linked. Another type of vector is a viral vector, such as an adeno-associated virus (AAV) vector, in which a foreign DNA segment is linked to the viral genome. Some vectors can replicate autonomously in host cells, while others can integrate into the host genome and replicate along with it.

[0131] "Host cell" and "cell line" are used interchangeably and refer to cells that have been introduced with exogenous nucleic acids and their offspring, regardless of the number of passages. Offspring are not allowed to be completely identical to the parent cells in terms of nucleic acids; they can contain mutations, as long as the offspring with mutations have the same desired function or activity as the primary cells.

[0132] "Subject" or "individual" refers to an animal, preferably a mammal. Depending on the specific implementation plan, the subject is a mammal, including, for example, a camel, horse, cow, pig, sheep, cat, dog, rabbit, rat, guinea pig, mouse, non-human primate, and human. In the specific implementation plan, the subject is a human.

[0133] It should be understood that when a range of values ​​is publicly mentioned, such as “A to B”, it is a concise way of writing that, although not every point value in the range is given, is regarded as the integers and decimals in the range being clearly disclosed.

[0134] "And / or", for example "A and / or B", should be understood as meaning "A and B" or "A or B".

[0135] "Optional" or "optionally" means that the events or circumstances described below may, but do not necessarily, occur. The singular forms "an" and "a" used in the specification and claims include their corresponding plural references unless otherwise clearly stated in the text.

[0136] The following are examples of practices implemented in this application and should not be construed as limiting the scope of the invention in any way.

[0137] Example

[0138] Materials and reagents:

[0139] (1) S2100 Anti-fluorescence attenuation mounting medium (Solarbio, China)

[0140] (2) 7105 microscope slide (Sail brand, China)

[0141] (3) D9542 DAPI (Sigma, USA)

[0142] (4) ab53554 GFAP antibody (Abcam, UK)

[0143] (5) ab104224 NeuN antibody (Abcam, UK)

[0144] (6) 3450s PSD95 antibody (CST, USA)

[0145] (7) ZI-9305 Immunohistochemistry Pen (Zhongshan Jinqiao, China)

[0146] (8) G1101 Paraformaldehyde (Wuhan Sewell Biotechnology Co., Ltd., China)

[0147] (9) HI1220 film roaster

[0148] (10) Y Maze (Beijing Youbi Biotechnology Center, China)

[0149] (11) ANY-Maze animal behavior analysis software (Stoelting, USA)

[0150] (12) Etho Vison XT Animal Movement Tracking System (Noldus, Netherlands)

[0151] (13) TSA Multiplex Immunohistochemistry Kit (Akfa, China) Reagent Preparation:

[0152] 1. Preparation of PBS buffer: Dissolve 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 in 800ml distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to make up to 1L.

[0153] 2. Preparation of blocking solution: Use PBST (PBS + 0.1% Tween 20) containing 1% BSA and 22.52 mg / mL glycine.

[0154] Data calculation:

[0155] Quantitative calculation of the correct rate of spontaneous alternation: Alternation rate = (Number of correct alternations / (Total number of entries in all three arms - 2) × 100%

[0156] Recognition Index (RI) = New object / (New object + Old object) × 100%

[0157] Statistical methods:

[0158] All statistical operations in this study were performed on GraphPad Prism (version 8, GraphPad Software, San Diego, CA) and ANY-maze (version 7.2, Stoelting).

[0159] i) One-way ANOVA is used to analyze the effect of an independent variable on the dependent variable across multiple level groups.

[0160] ii) One-sample t-test, used to compare the difference between the sample mean and the population mean.

[0161] iii) The two independent samples t-test is used to test whether the difference between the means of two unknown populations represented by the means of two samples is significant.

[0162] Example 1: Preparation of scFv antibody specifically binding to Aβ

[0163] 1. Experimental Procedure

[0164] 1) Plasmid preparation

[0165] The target gene fragment (SEQ ID NO: 12) was amplified by PCR and inserted into the co-expression vector (pCMV3-C-His-NCV). The sequence of the constructed vector was verified by sequencing, and the plasmid was amplified.

[0166] pCMV3-C-His-NCV (vector name)

[0167] 2) Cell culture and transfection

[0168] The plasmid and transfection reagent were mixed in the optimal ratio and added to a bioreactor containing HEK293 cells. The cells were cultured in serum-free medium and kept at 37°C for 6 days with appropriate stirring in an Erlenmeyer flask.

[0169] 3) Purification and Analysis

[0170] The cell culture medium was centrifuged, and the cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and elution with an appropriate buffer, the eluent was loaded onto a size exclusion chromatography column, and the purified protein was analyzed by SDS-PAGE.

[0171] 2. Experimental Results

[0172] Coomassie Brilliant Blue staining revealed that the target protein band was 30.65 kDa in non-denaturing gel and 31.09 kDa in denaturing gel, confirming that the isolated and purified protein was the designed and prepared antibody scFv-ANG (Figure 1).

[0173] Example 2: Affinity verification of scFv antibodies that specifically bind to Aβ

[0174] 1. Experimental Procedure

[0175] 1) Use PBS to dilute the antibody.

[0176] The antigen used in the experiment was an Aβ oligomer. 1 mg of Aβ42 powder was dissolved in cooled hexafluoroisopropanol (HFIP) and incubated at room temperature for 60 min to ensure complete dissolution of Aβ42. The Aβ42 peptide-HFIP was then placed on ice for 5-10 min before being transferred to a fume hood with the cap opened to allow the HFIP to evaporate. After air drying, a transparent Aβ42 peptide was formed, which was dissolved in 44 μL of fresh anhydrous 100% dimethyl sulfoxide to prepare a 5 mmol / L Aβ oligomer for subsequent experiments.

[0177] 2) Set up two sets of SSA sensors. The first set is a protein immobilization detection sensor, and the second set is a control blank sensor. Both sets of SSA sensors use the same batch of sensors. Pre-wet the four SSA sensors in PBS for more than 10 minutes in two columns. Two sensors are used to immobilize biotinylated proteins, and the other two sensors are used as reference sensors and do not need to be immobilized.

[0178] 3) Set the sample plate and experimental steps in the Experiment Wizard software and start the measurement.

[0179] 2. Experimental Results

[0180] The results of the affinity verification experiment are shown in Figure 2, and the obtained ka, kd and KD values ​​are shown in Table 1.

[0181] Table 1

[0182] Example 3: In vivo administration of scFv antibodies that specifically bind to Aβ

[0183] 5XFAD mice express human APP and PSEN1 transgenes, with a total of 5 AD-related mutations: Swedish (K670N / M671L), Florida (I716V), and London (V717I) familial Alzheimer's disease (FAD) mutations in APP, and two FAD mutations in PSEN1, M146L and L286V.

[0184] 5XFAD mice rapidly and significantly exhibit the core pathological features of Alzheimer's disease (AD) (Aβ plaques, neuronal loss, and neuroinflammation), highly consistent with the pathological process of human AD, making them suitable as a model for studying the pathological mechanisms of AD. Because 5XFAD mice carry multiple AD-related mutant genes, they can provide clear genotype-phenotype association data when studying the interaction between anti-Aβ monoclonal antibodies and AD pathology. The significant neuroinflammatory response in 5XFAD mice provides an excellent experimental platform for studying the mechanisms by which anti-Aβ monoclonal antibodies affect neuroinflammation. The rapid development of obvious AD pathological features in 5XFAD mice saves research time and costs compared to other animal models. Furthermore, 5XFAD mice not only share similar pathological features with human AD but also show similarities in drug response and biomarkers, making them a powerful tool for studying treatment and diagnosis methods for human AD.

[0185] 5XFAD mice were divided into three groups of 10 mice each. Starting from week 28, each group received a weekly intravenous injection via tail vein of either the Aβ-specific scFv antibody (scFv-ANG, concentration 2 mg / ml, dose 10 mg / kg), PBS, or aducunabumab (concentration 2 mg / ml, dose 10 mg / kg). After four weeks of administration, behavioral experiments were conducted on each group of mice, including novel object recognition, Y maze, and Morris water maze.

[0186] Example 4: Y-maze

[0187] The Y-maze can be used to assess short-term memory in mice. By allowing mice to explore all three arms of the maze, spontaneous alternation behavior—a measure of spatial working memory—can be assessed, driven by the rodent's innate curiosity to explore previously unvisited areas. A mouse with intact working memory, possessing full prefrontal cortex function, will remember previously visited arms and tend to enter the less recently visited arms.

[0188] 1. Experimental Procedure

[0189] i) Set up the equipment and video source, adjust the camera focal length and the position of the Y maze, use the multiline tool to outline the Y maze, delineate and close the experimental area, and use the green ruler to select the arms of known length.

[0190] ii) Set the animal color according to the shade of the animal color and the background color, and select the standard sensitivity.

[0191] iii) Set to track the animal's head and tail, select general settings.

[0192] iv) Define the zones and name them A, B, and C.

[0193] v) Set the sequence: Set 6 sequences, namely ABC, ACB, BAC, BCA, CAB, and CBA.

[0194] vi) Set the experiment duration to 8 minutes.

[0195] vii) Define the formula for calculating the spontaneous alternation rate: Alternation rate = (ABC + ACB + BAC + BCA + CAB + CBA) / (A + B + C - 2) * 100%

[0196] 2. Experimental Results

[0197] Figure 4B shows the total number of times the animals entered the maze arm. The total number of times the negative control group, scFv-ANG group, and adunatumab treatment group entered the arm were 27.10±7.752, 22.70±4.990, and 23.90±3.314, respectively. There was no statistically significant difference among the groups (p>0.05), indicating that this behavioral experiment can effectively reflect the differences in working memory of 5XFAD mice among different groups.

[0198] Figure 4C shows the changes in spontaneous alternation rate among different groups. The spontaneous alternation rate in the negative control group was 0.4245±0.1387. Compared with the negative control, the spontaneous alternation rate in the scFv-ANG group increased to 0.6641±0.0663 (p<0.0001), and the spontaneous alternation rate in the adunatumab treatment group was 0.5895±0.04968 (p=0.0014). The spontaneous alternation rate in the scFv-ANG group was higher than that in the adunatumab treatment group, reflecting that its spatial memory and working memory abilities were effectively enhanced compared with the negative control group. The working memory of the adunatumab group was also enhanced to a certain extent compared with the control group, but the degree was not as great as that of the scFv-ANG group.

[0199] Figure 4D shows the Y-maze trajectory diagrams for different drug administration groups. It can be found that the mice in the scFv-ANG treatment group entered arms A, B, and C approximately the same number of times, while the control group preferred to enter a specific arm.

[0200] Example 5: New Object Recognition

[0201] The Novel Object Recognition (NOR) test evaluates an animal's cognitive memory ability by measuring the time it takes for experimental animals to explore familiar objects and new, unfamiliar objects.

[0202] 1. Experimental steps i) Prepare a set of equipment for Novel Object Recognition (NOR): a square box and camera equipment.

[0203] ii) There are three objects, A, B and C. Objects A and B are completely identical, while object C is very different from both objects A and B. Its diameter is 3cm.

[0204] iii) Before conducting the test, eliminate the unfamiliarity between the mouse and touch the mouse every day to avoid stimulating the mouse during the operation.

[0205] Adaptation period: Mice were allowed to move freely inside the device for 10 minutes.

[0206] Acquaintance period: Place two identical objects in the device, 10cm away from both arms. Place the mouse with its back to the object from the same distance. Use a camera and software to record the time the mouse spends exploring each object (exploration is defined as the mouse's mouth or nose touching the object or getting within 2-3cm of the object). Measure the number of times, time and distance the animal explores each object within 5 minutes.

[0207] Testing period: One hour after the completion of the second stage, the memory was tested. One of the two identical objects was replaced with a different object and placed in the device. The mouse was placed in the device with its back to the object from an equal distance for 5 minutes.

[0208] 2. Experimental Results

[0209] Figure 5B shows the total number of explorations. The total number of arm insertions in the negative control group, scFv-ANG group, and adunatumab treatment group were 45.90±10.94, 53.10±13.54, and 55.90±10.74, respectively. There was no statistically significant difference among the groups (p>0.05), indicating that there was no significant difference in motor ability among the 5XFAD mice, and the experimental results can reflect the spatial memory ability of the mice.

[0210] Figure 5C illustrates the differences in cognitive indices among different groups. Mice with poor cognitive abilities showed no difference in exploring new and old objects; mice with normal cognitive abilities took longer to explore new objects than old ones. The cognitive index of the control group was 0.5188±0.08323. After treatment with scFv-ANG, the cognitive index of 5XFAD mice was significantly higher than that of the control group, increasing to 0.6042±0.04423 (p=0.0013), indicating that scFv-ANG can alleviate cognitive impairment in mice. The cognitive index of the aducunamumab treatment group was 0.5417±0.08047, with no statistically significant difference from the control group, indicating that scFv-ANG antibody was more effective than aducunamumab in improving spatial cognition in 5XFAD mice.

[0211] Figure 5D shows a heatmap of 5XFAD mice exploring objects. Compared to the control group, there was no significant difference in the time and number of times the mice explored new and old objects. After treatment with scFv-ANG, 5XFAD mice explored new objects more frequently and for longer periods than those exploring old objects. However, the number of times and the time spent exploring new objects in the 5XFAD mice treated with aducuna were not as high as those in the scFv-ANG treatment group.

[0212] Example 6: Morris Water Maze

[0213] 1. Experimental Procedure

[0214] A circular platform with a diameter of 10cm was placed on one side of the pool, and its position could be changed. The camera was positioned 2m above the center of the pool throughout the experiment, ensuring the water temperature was maintained between 22℃ and 24℃. The water maze was divided into four directions according to NSWE, forming four quadrants: NW, NE, SW, and SE. The platform was fixed in any quadrant, and its position remained unchanged during training. The experiment consisted of the following three phases:

[0215] 1) Phase 1 (Cue Test, Day 1)

[0216] The primary objective of the first phase is to assess the animals' vision and mobility, and to help them develop a "platform" concept. A one-day cue test is conducted. Water is poured into the pool, and titanium dioxide is used to whiten the water, making it easier to locate the mice. A conspicuous marker approximately 12cm high is fixed to the platform. Mice are placed in the four quadrants NW, NE, SW, and SE, ensuring their heads face the pool wall. The experiment ends when a mouse swims onto the platform or after a 60-second timeout. If a mouse fails to swim onto the platform within 60 seconds, it is guided to the platform and allowed to remain there for 15 seconds before being returned to its cage. Each mouse must be at least 30 minutes apart from each experiment, and all mice must complete the experiment at a specific platform location before moving the platform to the next quadrant to continue.

[0217] 2) Second stage (acquisition period, days 2-4)

[0218] During this phase, remove the prominent markers from the platform, keeping it 1 cm underwater. This ensures the mouse cannot see the platform while allowing it to use buoyancy to climb onto it. The platform's position must remain unchanged throughout the training phase. Place four different, prominent directional markers around the pool to provide environmental cues for the mouse.

[0219] Mice were placed into the water in different directions in sequence. The experiment was stopped when the mice swam onto the platform or after 60 seconds. Similarly, if the mice failed to swim onto the platform on their own within 60 seconds, they were guided to the platform and allowed to stay there for 15 seconds before being returned to their cages. Four training cycles were conducted each day. During each training cycle, mice were placed into the water from different locations, and their movement trajectories and latency to reach the platform were recorded.

[0220] 3) Phase Three (Probe Test, Day 5)

[0221] Remove the platform, set each experiment to last 60 seconds and then automatically end, place the mice on the opposite side of the platform, and record the movement trajectories of all mice during this period, as well as the number of times and time spent traversing the target quadrant / original platform.

[0222] 2. Experimental Results

[0223] Figure 6B shows the trajectory diagrams between different drug administration groups in Test 1 and Test 2. Test 1 is a navigational test used to measure the mice's ability to learn and remember a water maze. The experiment observed and recorded the route the mice took to find and climb the platform, as well as the time required, i.e., recording the latency period. The results showed that the 5XFAD group treated with scFv-ANG had the shortest path and time to find the platform, reflecting improved spatial memory. Test 2 is a spatial probe test used to measure the mice's ability to retain the spatial location memory of the platform after learning to find it. After the navigational test, the platform was removed, and the mice were placed back into the water from the same entry point. The time it took to reach the original platform location for the first time and the number of times they crossed the original platform were measured. The results showed that mice treated with scFv-ANG crossed the original platform more times.

[0224] Figure 6C shows the changes in swimming speed of mice under different training days. The average speed of the control group over 5 days was 0.1914±0.04942 m / s, the average swimming speed of mice in the scFv-ANG treatment group over 5 days was 0.1809±0.03153 m / s, and the average swimming speed of mice in the aducanamab treatment group over 5 days was 0.1949±0.02706 m / s. It can be seen that the average swimming speed of each group did not change over different training days (p>0.05), and there was no difference in the exercise ability of each group or between groups, thus excluding the error caused by changes in exercise ability.

[0225] Figure 6D shows the time it took for 5XFAD mice to reach the platform in different treatment groups. The time to reach the platform in the control group was 9.6±4.6s, while the time to reach the platform in mice treated with scFv-ANG antibody was significantly shortened (p=0.0094), to 3.8±2.394s. The time to reach the platform in 5XFAD mice treated with aducanamab was also shortened to 7.4±4.719s, which was not statistically significant compared with the negative control (p>0.05). This indicates that scFv-ANG has a stronger effect on restoring spatial memory in 5XFAD mice.

[0226] Figure 6E shows the number of times the animal crossed the platform after its removal. The number of platform crossings is an important indicator in spatial search experiments; it represents the number of times the experimental animal traverses the original platform location within a certain time after the platform is removed. The more times an animal traverses the original platform location, the better its spatial learning and memory ability. The control group had 0.800±0.9189 platform crossings, while the mice treated with scFv-ANG showed a significant increase to 2.300±1.418 platform crossings after platform removal (p=0.0317), indicating a recovery in their spatial learning and memory ability. The 5XFAD mice in the aducunamumab treatment group also showed an increase to 2.600±1.350 platform crossings, a significant improvement compared to the control group (p=0.009), indicating that treatment with anti-Aβ antibodies (scFv-ANG antibody or aducunamumab) has a significant alleviating effect on spatial memory ability in mice.

[0227] Example 7: Multicolor immunofluorescence

[0228] 1. Experimental Procedure

[0229] 5XFAD mice were divided into 3 groups of 10 mice each. Starting from week 28, each group was injected weekly via tail vein with the specific Aβ-binding scFv antibody (concentration 2 mg / ml, dose 10 mg / kg), PBS, or aducanamab (concentration 2 mg / ml, dose 10 mg / kg). After 4 weeks of administration, brain tissue samples were taken, paraffin sections were prepared, and multicolor immunofluorescence experiments were performed.

[0230] (1) Brain tissue sampling

[0231] i) Anesthesia: 0.7% sodium pentobarbital was injected intraperitoneally into the mice to be sampled at a dose of 50 mg / kg. The mice lost the ability to move their limbs 5-10 minutes after the injection. After confirming that the mice were normal, the next step was carried out.

[0232] ii) Fixation: Fix the mouse limbs on a pre-sterilized foam board and disinfect the mouse abdomen and head with 75% ethanol.

[0233] iii) Prepare two brand new disposable 20mL syringes. Fill one syringe with 1xPBS and the other with 4% PFA fixative. Set aside for later use.

[0234] iv) Dissection: Use tweezers to grasp the abdominal skin, cut the hair and extend it longitudinally to the chest cavity, cut open the chest cavity and ribs to expose the liver and heart.

[0235] v) Perfusion: The right atrial appendage of the mouse was cut open, and a syringe containing 1×PBS was inserted into the left ventricle (slightly to the right and below the apex of the heart). The 1×PBS solution was slowly perfused into the heart. Observing the liver, spleen, and kidneys, etc., and observing their grayish-white color indicates normal perfusion. After completion, a syringe containing 4% PFA fixative was inserted into the same insertion site in the left ventricle, and 20 mL was slowly perfused into the mouse. Observing the mouse's limbs and tail, rigidity was observed, or the mouse's tail was touched; rigidity indicates good perfusion effect.

[0236] vi) Brain harvesting: After perfusion, the mouse head was removed. First, the scalp was cut open from both sides, then the skull along the line connecting the two sides of the eyeballs and the skull on the upper side of the brainstem were cut open to completely separate the brain tissue. The brain was symmetrically cut into left and right hemispheres along the midline of the brain with a scalpel. The left hemisphere was placed in a 15mL centrifuge tube containing 4% PFA fixative, and the right hemisphere was placed in a cryovial with the corresponding number. The hemispheres were first flash-frozen in liquid nitrogen and then transferred to a freezer at -80°C.

[0237] (2) Tissue sections

[0238] 1) Dehydration: After the brain tissue was fixed at 4°C for 24 hours, it was dehydrated by preparing 30% sucrose with 1xPBS. The left hemisphere of the brain tissue was dehydrated. The dehydration process was carried out at room temperature, with rotation throughout to ensure sufficient fluid replacement, and lasted for 48 hours.

[0239] 2) Paraffin-embedded sections: The brain tissue that was being fixed was then embedded in paraffin.

[0240] (3) Multicolor immunofluorescence

[0241] 1) Select the preserved paraffin sections in order and place them on the slide rack.

[0242] 2) Baking: Baking the film at 60°C for 15 minutes on a baking machine, and then immediately transferring it to a dewaxing solution for dewaxing.

[0243] 3) Dewaxing: Rinse in the following order: dewaxing solution 1, dewaxing solution 2, anhydrous ethanol 1, anhydrous ethanol 2, 75% ethanol, 50% ethanol, and double distilled water, for 10 minutes each time.

[0244] 4) Antigen retrieval: Place the slide in a pressure cooker, heat it to boiling on the maximum power of the induction cooker, then turn to low heat and continue boiling for 8 minutes, and then let it cool naturally to room temperature.

[0245] 5) Change the washing box, rinse once with pure water, rinse once with 1×PBS, 2 min.

[0246] 6) Endogenous HRP inactivation: After antigen retrieval in tissue sections, add sufficient 3% hydrogen peroxide stock solution and incubate at room temperature for 15 min.

[0247] 7) Rinse twice with 1xPBS, 3 min each time. After incubation with hydrogen peroxide stock solution, draw hydrophobic circles around the tissue.

[0248] 8) Blocking: Add sufficient blocking solution and incubate at room temperature for 10 minutes.

[0249] 9) Incubate with primary antibody, and dilute the primary antibody with blocking buffer.

[0250] 10) Rinse three times with 1×PBS, 2 min each time.

[0251] Repeat steps 4-10, staining sequentially (Aβ, NeuN, GFAP, p-tau).

[0252] 11) Counterstain with DAPI for 30 minutes.

[0253] 12) Rinse once with 1×PBS, then rinse once with pure water for 2 min.

[0254] 13) Cover the film.

[0255] 2. Experimental Results

[0256] Figure 7A shows the immunofluorescence staining of Aβ and GFAP in the hippocampus and cortical regions. The results show that after treatment with scFv-ANG and adunamoba, the number of Aβ plaques was significantly reduced, and the fluorescence intensity of GFAP, an important indicator of neuroinflammation, was also significantly lower than that of the control group, indicating that the level of neuroinflammation was improved.

[0257] Figures 7B to 7E show the quantitative statistics of plaque burden, including the number and diameter of plaques. The average number of plaques in the cortex of the control group 5XFAD mice was 134.4 ± 19.375. After treatment with scFv-ANG antibody, the number of plaques significantly decreased (p < 0.001) to 78.3 ± 6.2. Similarly, after treatment with aducunabumab, the number of plaques in the cortex of 5XFAD mice also significantly decreased (p < 0.001) to 84.4 ± 24.4. In the hippocampus, the average number of plaques in the control group was 92.8 ± 14.06. After treatment with scFv-ANG antibody, the number of plaques in the hippocampus of 5XFAD mice decreased to 51.04 ± 4.7. After treatment with aducunabumab, the number of plaques in the hippocampus of 5XFAD mice also decreased to 60.87 ± 18.73. It was observed that the Aβ load in 5XFAD mice was significantly improved after administration of scFv-ANG antibody (p<0.05), decreasing from 662.5±190.6 in the control group to 293.8±93.8. Simultaneously, the plaque diameter after treatment with scFv-ANG antibody and aducunabumab was significantly reduced compared to the control group's 31.6±3.75 μm (p<0.005), decreasing to 8.5±2.6 μm and 15.1±3.56 μm, respectively.

[0258] Figure 7F shows the Aβ plaque staining pattern in the sagittal plane of the whole brain and a magnified view of the hippocampus. After treatment with scFv-ANG, the Aβ plaque burden in the whole brain and hippocampus of 5XFAD mice was significantly reduced, and the number and size of the plaques were decreased, indicating that the scFv-ANG antibody can effectively reduce the Aβ burden.

Claims

1. An Aβ-specific scFv antibody, wherein the Aβ-specific scFv antibody comprises, from 5' to 3', the following components: Heavy chain variable region (VH), Linking peptides Light chain variable region (VL) and 3' terminal sequence, in: The heavy chain variable region includes the following complementary determinant region (CDR): The amino acid sequence of HCDR1 is shown in SEQ ID NO:

1. The amino acid sequence HCDR2 is shown in SEQ ID NO:

2. The amino acid sequence is HCDR3 as shown in SEQ ID NO: 3; The light chain variable region includes the following complementary determinant region (CDR): The amino acid sequence of LCDR1 is shown in SEQ ID NO:

4. The amino acid sequence LCDR2 is shown in SEQ ID NO:

5. The amino acid sequence is LCDR3 as shown in SEQ ID NO: 6; The CDR is based on the Kabat numbering rules; The amino acid sequence of the linker peptide is shown in SEQ ID NO: 9; and The amino acid sequence of the 3' terminal sequence is shown in SEQ ID NO:

10.

2. The scFv antibody that specifically binds to Aβ according to claim 1, wherein: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, or has at least 70% sequence identity with SEQ ID NO: 7; The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8, or has at least 70% sequence identity with SEQ ID NO:

8. Preferably, the full-length amino acid sequence of the scFv antibody that specifically binds to Aβ is shown in SEQ ID NO:

11.

3. A pharmaceutical composition comprising: Effective amounts of the scFv antibody that specifically binds to Aβ according to claim 1 or 2, for prevention or treatment. One or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

4. The pharmaceutical composition according to claim 3, wherein: The pharmaceutical composition is in liquid, semi-solid, and solid dosage form, preferably in liquid solution, suspension, tablet, pill, powder, liposome, or suppository, and more preferably in injectable or infusionable solution; Preferably, the pharmaceutical composition is administered via parenteral route, such as intravenous, subcutaneous, intraperitoneal, or intramuscular injection, more preferably via intravenous administration.

5. An isolated nucleic acid encoding an scFv antibody that specifically binds to Aβ as described in claim 1 or 2.

6. An expression vector comprising the isolated nucleic acid according to claim 5.

7. A host cell comprising the isolated nucleic acid according to claim 5 or the expression vector according to claim 6. Preferably, the host cell is a prokaryotic or eukaryotic host cell, more preferably a eukaryotic host cell, such as mammalian cells, insect cells, plant cells and fungal cells, and even more preferably mammalian cells, such as human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells.

8. Use of the scFv antibody that specifically binds to Aβ as described in claim 1 or 2, the pharmaceutical composition as described in claim 3 or 4, or the isolated nucleic acid as described in claim 5 in the preparation of a medicament, wherein: The drug is used for any one or a combination of the following: Prevent or treat Alzheimer's disease; reduce Aβ plaque burden, number of plaques, and plaque diameter; improve spatial memory and cognitive function.

9. A non-diagnostic method for determining Aβ levels in a sample in vitro, comprising the following steps: The sample is exposed to an effective amount of the scFv antibody that specifically binds to Aβ as described in claim 1 or 2.

10. A kit comprising at least one container, each container independently containing: The scFv antibody that specifically binds to Aβ as described in claim 1 or 2, the pharmaceutical composition as described in claim 3 or 4, the isolated nucleic acid as described in claim 5, the expression vector as described in claim 6, or the host cell as described in claim 7; The kit is used to diagnose or detect Aβ protein in samples or cells expressing Aβ protein; Preferably, the kit contains one or more of the following components: Analytical reagents Buffer solution, The scFv antibody that specifically binds to Aβ as described in claim 1 or 2, and Optional: Instruction manual.

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