Anti-aβ antibody, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of antibody drugs, and in particular, to an anti-N3pE amyloid-β antibody and the use thereof, and a pharmaceutical composition containing the anti-N3pE amyloid-β antibody. The anti-N3pE amyloid-β antibody exhibits a significantly excellent affinity, stability and specificity. In addition, further provided is the use of the anti-N3pE amyloid-β antibody for treating diseases caused by amyloid-β (e.g., Alzheimer's disease, Down syndrome, cerebral amyloid angiopathy, etc.).

Description

Anti-Aβ antibodies, their preparation methods and applications

[0001] This application claims priority to Chinese patent application No. 202510125837.7, filed on January 27, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of antibody drug technology, and more specifically, to humanized anti-Aβ monoclonal antibodies, their preparation methods, and applications. Background Technology

[0003] Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by the deposition of β-amyloid-β (Aβ) plaques and the formation of neurofibrillary tangles in the brain. Numerous studies have shown that abnormal aggregation and deposition of Aβ play a crucial role in the pathogenesis of AD, particularly its soluble oligomeric form, which is considered neurotoxic and capable of inducing neuroinflammation, synaptic dysfunction, and neuronal death.

[0004] Therefore, antibody therapy targeting Aβ has become one of the important research directions in the field of AD treatment in recent years. By developing monoclonal antibodies that can efficiently recognize and clear specific forms of Aβ, it is hoped that the pathological progression of AD can be slowed or halted. The antibodies that are progressing most rapidly in clinical development include Bapineuzumab, Solanezumab, Crenzumab, Gantenerumab, Aducanumab, Lecanemab, and Donanemab. These antibodies recognize different antigenic sites on Aβ, and they vary in their ability to significantly reduce plaque burden. All of these antibodies have been tested in phase III studies in cohorts of patients with mild cognitive impairment or mild AD dementia. Based on previous comparisons of Aβ-PET signaling and post-mortem Aβ burden, Aβ deposition may be reduced accordingly. To date, the three antibodies with the highest Aβ deposition removal rates (>60% after 18 months of treatment) have been identified—Lecanemab, Donanemab, and Aducanumab. A common feature of these antibodies in clearing Aβ deposition in clinical practice is that they recognize N-terminal amino acids, epitopes exposed on mature amyloid fibrils. In contrast, Solanezumab and Crenzumab only recognize intermediate sequence epitopes embedded within amyloid fibrils, thus primarily recognizing monomeric Aβ. Donanemab and Aducanumab, the two antibodies most effective at removing Aβ from the brain, primarily recognize large amyloid aggregates.

[0005] However, due to the limitations of current antibody development technology, the development of Aβ antibodies still faces many challenges, such as improving their specificity, reducing immunogenicity, and optimizing brain delivery efficiency. Therefore, designing Aβ antibody compounds with superior properties is of great significance for advancing the treatment of Alzheimer's disease (AD). Summary of the Invention

[0006] This disclosure aims to discover Aβ antibodies with stronger plaque clearance capabilities, with the goal of developing more effective AD drugs than those currently on the market. To address the problems existing in the prior art, the purpose of this disclosure is to provide a humanized anti-Aβ monoclonal antibody or its antigen-binding fragment, said antibody or its antigen-binding fragment specifically binding to Aβ [N3pE] and not significantly binding to Aβ [WT]. It also provides a vector, host cell, and uses for encoding the nucleotides of this monoclonal antibody. The sequence of the variable region of the antibody gene provided in this disclosure can be used to construct a full-length antibody molecule for clinical use as a drug to treat and diagnose amyloidosis-related diseases and conditions (such as Alzheimer's disease).

[0007] To achieve the above-mentioned objectives, this disclosure provides the following technical solution:

[0008] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that binds to amyloid β (Aβ), comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3.

[0009] The amino acid sequence of HCDR1 is selected from SEQ ID NO:1, 9, 17 and 25;

[0010] The amino acid sequence of HCDR2 is selected from SEQ ID NO:2, 10, 18 and 26;

[0011] The amino acid sequence of HCDR3 is selected from SEQ ID NO:3, 11, 19 and 27;

[0012] The amino acid sequence of LCDR1 is selected from SEQ ID NO:4, 12, 20 and 28;

[0013] The amino acid sequence of LCDR2 is selected from SEQ ID NO:5, 13, 21 and 29; and / or

[0014] The amino acid sequence of LCDR3 is selected from SEQ ID NO:6, 14, 22 and 30.

[0015] In some embodiments, the amino acid sequence of its heavy chain variable region is selected from SEQ ID NO:7, 15, 23 and 31; and / or the amino acid sequence of its light chain variable region is selected from SEQ ID NO:8, 16, 24 and 32.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 of the heavy chains and LCDR1, LCDR2, and LCDR3 of the light chains, as shown in any one of the following groups:

[0017] (1) HCDR1 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, HCDR3 contains the amino acid sequence shown in SEQ ID NO:3, LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

[0018] (2) HCDR1 contains the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains the amino acid sequence shown in SEQ ID NO:10, HCDR3 contains the amino acid sequence shown in SEQ ID NO:11, LCDR1 contains the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:14.

[0019] (3) HCDR1 contains the amino acid sequence shown in SEQ ID NO:17, HCDR2 contains the amino acid sequence shown in SEQ ID NO:18, HCDR3 contains the amino acid sequence shown in SEQ ID NO:19, LCDR1 contains the amino acid sequence shown in SEQ ID NO:20, LCDR2 contains the amino acid sequence shown in SEQ ID NO:21, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:22.

[0020] (4) HCDR1 contains the amino acid sequence shown in SEQ ID NO:25, HCDR2 contains the amino acid sequence shown in SEQ ID NO:26, HCDR3 contains the amino acid sequence shown in SEQ ID NO:27, LCDR1 contains the amino acid sequence shown in SEQ ID NO:28, LCDR2 contains the amino acid sequence shown in SEQ ID NO:29, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:30.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises HCDR of the heavy chain and LCDR of the light chain, as shown in any of the following groups:

[0022] (1) HCDR1 of the amino acid sequence shown in SEQ ID NO:1, HCDR2 of the amino acid sequence shown in SEQ ID NO:2, HCDR3 of the amino acid sequence shown in SEQ ID NO:3, LCDR1 of the amino acid sequence shown in SEQ ID NO:4, LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:6.

[0023] (2) HCDR1 of the amino acid sequence shown in SEQ ID NO:9, HCDR2 of the amino acid sequence shown in SEQ ID NO:10, HCDR3 of the amino acid sequence shown in SEQ ID NO:11, LCDR1 of the amino acid sequence shown in SEQ ID NO:12, LCDR2 of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 of the amino acid sequence shown in SEQ ID NO:14.

[0024] (3) HCDR1 of the amino acid sequence shown in SEQ ID NO:17, HCDR2 of the amino acid sequence shown in SEQ ID NO:18, HCDR3 of the amino acid sequence shown in SEQ ID NO:19, LCDR1 of the amino acid sequence shown in SEQ ID NO:20, LCDR2 of the amino acid sequence shown in SEQ ID NO:21, and LCDR3 of the amino acid sequence shown in SEQ ID NO:22.

[0025] (4) HCDR1 of the amino acid sequence shown in SEQ ID NO:25, HCDR2 of the amino acid sequence shown in SEQ ID NO:26, HCDR3 of the amino acid sequence shown in SEQ ID NO:27, LCDR1 of the amino acid sequence shown in SEQ ID NO:28, LCDR2 of the amino acid sequence shown in SEQ ID NO:29, and LCDR3 of the amino acid sequence shown in SEQ ID NO:30.

[0026] In some embodiments, the antibody or its antigen-binding fragment binds to Aβ in the form of N3pE; preferably, the antibody or its antigen-binding fragment does not bind to monomeric wild-type Aβ, and / or, the antibody or its antigen-binding fragment substantially does not bind to polymeric wild-type Aβ.

[0027] In some embodiments, the antibody has any one of the following sets of heavy chain variable region sequences and light chain variable region sequences:

[0028] (1) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:8; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:8;

[0029] (2) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:15, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:16; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:15 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:16;

[0030] (3) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:23, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:24; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:23 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:24;

[0031] (4) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:31, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:32; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:31 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:32.

[0032] In some embodiments, the antibody also has one or more of the following characteristics:

[0033] (1) It further includes a heavy chain constant region and / or a light chain constant region;

[0034] (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody;

[0035] (3) It is a monoclonal antibody;

[0036] (4) It is a full-length antibody, or it is a Fab, Fv, scFv, F(ab')2, linear antibody or single-domain antibody;

[0037] (5) It is in the form of IgG1, IgG2, IgG3 or IgG4;

[0038] (6) It binds to the form of polymer N3pE with an affinity of less than 20 nM.

[0039] In some embodiments, the heavy chain constant region comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:33; and the light chain constant region comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:34.

[0040] Preferably, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:33; the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:34;

[0041] Preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:33; and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:34.

[0042] In some implementations, each CDR area is defined according to the Kabat definition scheme, the Chothia definition scheme, the Abm definition scheme, the IMGT definition scheme, and / or the Contact definition scheme.

[0043] In another aspect of this disclosure, a fusion protein is provided, wherein one of the fused portions comprises the aforementioned antibody.

[0044] In another aspect of this disclosure, a bispecific antibody or a multispecific antibody is provided, wherein one antigen-binding domain comprises the aforementioned antibody or its antigen-binding fragment.

[0045] Another aspect of this disclosure provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described above.

[0046] Another aspect of this disclosure provides an expression vector comprising a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described above.

[0047] Another aspect of this disclosure provides a host cell that is transformed or transfected with the expression vector described above.

[0048] In another aspect of this disclosure, a method for preparing an antibody or an antigen-binding fragment thereof is provided, comprising culturing the host cells under conditions suitable for the production of the antibody or the antigen-binding fragment thereof; and isolating the antibody or the antigen-binding fragment thereof from the host cells and / or culture.

[0049] In another aspect of this disclosure, an antibody-drug conjugate is provided, comprising the aforementioned antibody or its antigen-binding portion, or the aforementioned bispecific or multispecific antibody, covalently attached to a therapeutic portion; preferably, the therapeutic portion is selected from cytotoxic drugs, cytokines, immunosuppressants, immunostimulants, cleaved peptides, and radioisotopes.

[0050] In another aspect of this disclosure, a pharmaceutical composition is provided comprising the antibody or its antigen-binding fragment, the fusion protein, the bispecific antibody or multispecific antibody, the nucleic acid molecule, the expression vector, the host cell or the antibody-drug conjugate, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0051] In another aspect of this disclosure, there are provided the above-mentioned antibody or its antigen-binding fragment, the above-mentioned fusion protein, the above-mentioned bispecific antibody or multispecific antibody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host cell, the above-mentioned antibody-drug conjugate or the above-mentioned drug composition, which are used for diagnosing or treating diseases related to Aβ aggregation and / or deposition in the brain of a subject;

[0052] Preferably, the diseases associated with Aβ accumulation and / or deposition in the subject's brain include amyloidosis and neurological diseases; more preferably, the amyloidosis includes secondary amyloidosis and age-related amyloidosis; and / or, the neurological disease is Alzheimer's disease, more preferably Alzheimer's disease-related mild cognitive impairment, mild Alzheimer's disease-related dementia, and familial early-onset Alzheimer's disease.

[0053] In another aspect of this disclosure, the use of the antibody or antigen-binding fragment thereof described above, the fusion protein described above, the bispecific antibody or multispecific antibody described above, the nucleic acid molecule described above, the expression vector described above, the host cell described above, the antibody-drug conjugate described above, or the pharmaceutical composition described above in the preparation of a medicament for diagnosing or treating diseases associated with Aβ aggregation and / or deposition in the brain of a subject is provided.

[0054] Preferably, the diseases associated with Aβ aggregation and / or deposition include amyloidosis and neurological diseases; more preferably, the amyloidosis includes secondary amyloidosis and age-related amyloidosis; and / or, the neurological disease is Alzheimer's disease, more preferably Alzheimer's disease-related mild cognitive impairment, mild Alzheimer's disease-related dementia, and familial early-onset Alzheimer's disease.

[0055] Another aspect of this disclosure provides a method for diagnosing or treating a disease associated with Aβ accumulation and / or deposition in the brain of a subject, comprising administering to a subject in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described above, a fusion protein as described above, a bispecific antibody or multispecific antibody as described above, a nucleic acid molecule as described above, an expression vector as described above, a host cell as described above, an antibody-drug conjugate as described above, or a pharmaceutical composition as described above.

[0056] Preferably, the diseases associated with Aβ accumulation and / or deposition in the subject's brain include amyloidosis and neurological disorders; more preferably, the amyloidosis includes secondary amyloidosis and age-related amyloidosis; and / or, the neurological disorder is Alzheimer's disease, more preferably Alzheimer's disease-related mild cognitive impairment, mild Alzheimer's disease-related dementia, and familial early-onset Alzheimer's disease.

[0057] In another aspect of this disclosure, a kit is provided, comprising an antibody or antigen-binding fragment thereof as described above, a fusion protein as described above, a bispecific antibody or multispecific antibody as described above, a nucleic acid molecule as described above, an expression vector as described above, a host cell as described above, an antibody-drug conjugate as described above, or a pharmaceutical composition as described above. Attached Figure Description

[0058] Figure 1 illustrates the binding of the four humanized antibodies, mIgG2a, Lecanemab, and Donanemab of this disclosure to various forms of Abeta.

[0059] Figure 2 shows the total abeta level in the right brain of 6-month-old APP / PS1 mice 12 weeks after administration.

[0060] Figure 3 shows the binding of Bh39A and Donanemab to the monomer and polymer abeta.

[0061] Figure 4 shows the staining of Donanemab and Bh39A in tissue sections.

[0062] Figure 5 shows the microscopic staining image of a section at the median area (% of the area).

[0063] Figure 6 shows the Bruce Blue staining patterns in each group.

[0064] The abbreviations Aβ and N3pE-Aβ are Aβ proteins with pyroglutamylation at the N-terminus (N3pGlu-Aβ, where Glu can be abbreviated as E). CBS is carbonate buffer. Detailed Implementation

[0065] I. Definition

[0066] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0067] The terms "diseases associated with the accumulation and / or deposition of Aβ (e.g., N3pE Aβ) in the brain of a subject" or "diseases associated with Aβ peptide activity" refer to diseases pathologically characterized by Aβ deposits in the brain or cerebral vascular system. This includes diseases such as Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy. The clinical diagnosis, stage, or progression of Alzheimer's disease can be readily determined by a primary diagnosing physician or healthcare professional skilled in the art using known techniques and by observational findings. This typically includes brain plaque imaging, mental or cognitive assessments (e.g., Clinical Dementia Rating-Box Sum (CDR-SB), Mini-Mental State Examination (MMSE), or Alzheimer's Disease Assessment Scale-Cognitive (ADAS-Cog)) or functional assessments (e.g., Alzheimer's Disease Cooperative Study - Activities of Daily Living (ADCS-ADL)). Cognitive and functional assessments can be used to determine changes in a patient's cognition (e.g., cognitive decline) and function (e.g., functional decline). A reduction or slowing of cognitive decline can be measured by cognitive assessments such as the Clinical Dementia Rating-Box Sum, Mini-Mental State Examination, or Alzheimer's Disease Assessment Scale-Cognitive. A reduction or slowing of functional decline can be measured by functional assessments such as ADCS-ADL.

[0068] In one aspect, this document provides antibodies (e.g., monoclonal antibodies) that specifically bind to N3pE Aβ (or N3pGlu Aβ) and their antigen-binding fragments, or antibodies and their antigen-binding fragments that are substantially identical thereto. In a specific aspect, this document provides monoclonal antibodies that specifically bind to human N3pE Aβ, wherein said anti-N3pE Aβ antibodies comprise variants of the parent antibody. In a specific aspect, this document provides antibodies that specifically bind to N3pE Aβ (e.g., human N3pE Aβ). In a particular aspect, this document provides anti-N3pE Aβ containing modifications in one or more amino acid residues (e.g., 5-13 amino acid substitutions in the framework region of the heavy chain variable region) that maintain affinity for the antigen compared to parent antibodies without said modifications.

[0069] Regarding antibody chain polypeptide sequences, the phrase "substantially identical" can be understood as an antibody chain exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference polypeptide sequence. Regarding nucleic acid sequences, the term can be understood as a nucleotide sequence exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference nucleic acid sequence.

[0070] The term "sequence identity" or "identity" has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of the polynucleotide or polypeptide or along a region of the molecule. Although many methods exist for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0071] Regarding the variable domains of antibodies, the term "variable" refers to certain portions of related molecules with extensive sequence differences between antibodies, used for the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the variable domains of an antibody. Variability is concentrated in three segments known as complementarity-determining regions (CDRs; namely CDR1, CDR2, and CDR3) or hypervariable regions, all located within the variable domains of the light and heavy chains. More conserved portions within the variable domains are called framework (FR) regions or framework sequences. Each variable domain of the natural heavy and light chains comprises four FR regions, primarily employing a β-sheet configuration, linked by three CDRs forming loops that connect the β-sheet structure and, in some cases, partially form a β-sheet. The CDRs of each chain are typically linked in proximity by the FR regions, and the formation of antibody target binding sites (epitopes or determinants) is facilitated by CDRs from other chains. As used herein, immunoglobulin amino acid residue numbering follows the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise stated. A CDR may have the ability to specifically bind associated epitopes.

[0072] As used herein, an "antibody fragment" or "antigen-binding fragment" refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the antibody's variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity and at least a portion of the full-length antibody's specific binding ability. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments. The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that acquires immune-specific binding (i.e., exhibits at least or at least about 10) when inserted into an antibody frame (e.g., by replacing the corresponding region). 7 -10 8 M -1 Antibodies against the Ka antigen. A “functional fragment” or “antibody analogue against tight junction protein 18.2” is a fragment or analogue that prevents or substantially reduces the ability of said receptor to bind ligands or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as “antibody fragment,” and in the context of antibodies, it can refer to a fragment that prevents or substantially reduces the ability of said receptor to bind ligands or initiate signal transduction, such as F… v F ab F (ab′)2 Wait a minute. "F" v "A fragment is a dimer (V) formed by the non-covalent bonding of a variable domain of a heavy chain and a variable domain of a light chain." H -V L (Dimer) composition. In this configuration, the three CDRs of each variable domain interact to determine V. H -V L The target binding sites on the surface of the dimer are the same as in the case of the intact antibody. The six CDRs collectively confer target binding specificity to the intact antibody. However, even a single variable domain (or F containing only 3 target-specific CDRs) can affect the specificity of the target binding site. v (Half of it) can still have the ability to identify and bind to targets.

[0073] As used herein, “monoclonal antibody” refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0074] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells. As is known to those skilled in the art, hybridomas can proliferate and continuously supply the production of specific monoclonal antibodies. Methods for generating hybridomas are known in the art. When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0075] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies with the same domains.

[0076] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0077] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0078] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this disclosure also cover antibodies containing one or two amino acid mutations within the CDRs.

[0079] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody binds at its complementary site. Epitope determinants typically comprise chemically active surface subtypes of a molecule, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features as well as specific charge characteristics.

[0080] As used herein, the terms “specific binding” and “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of approximately 1 × 10⁻⁶. 7 M -1 Or 1x10 8 M -1 Or a larger affinity constant Ka (or 1x10) -7 M or 1×10 -8 The affinity constant (M or lower) binds to the antigen. The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art; see also U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating antibody binding affinity. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available.

[0081] As used herein, the term "nucleic acid molecule" refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0082] Also provided are “conserved sequence modifications” of the sequences listed herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of antibodies to antigens encoded by nucleotide sequences or containing amino acid sequences. These conserved sequence modifications include conserved nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved sequence modifications include conserved amino acid substitutions, wherein amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues in the anti-tight junction protein 18.2 antibody are preferably replaced by another amino acid residue from the same side chain family. Methods for identifying nucleotides and conserved amino acid substitutions that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0083] As an alternative, in another embodiment, mutations can be randomly introduced along all or part of the Aβ antibody coding sequence, for example, through saturation mutagenesis, and the resulting modified Aβ antibodies can be screened for improved binding activity.

[0084] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0085] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0086] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0087] As used herein, “treatment” for an individual suffering from a disease or disease condition means that the individual’s symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0088] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0089] As used in this article, the term "subject" refers to a mammal, such as a human.

[0090] Example

[0091] Example 1: Construction of a stable monoclonal cell line

[0092] 1.1 Preparation of antigen

[0093] Antigen design used the Aβ[N3pe] target for screening, and P7[N3pe] was designed. 3-7 P8[N3pe] 3-8 P9[N3pe] 3- Nine antigenic peptides were coupled to KLH / BSA via cysteine ​​residues and used as antigens for subsequent mouse immunization. The peptide sequences of the antigens used in this experiment include:

[0094] 1.2 Hybridoma Fusion Screening

[0095] The materials and reagents used were: experimental adjuvant (Sigma Adjuvant System, LOT#059M4170V, purchased from Sigma); PBS solution (PBS pH 7.2 basic (1×), LOT#8119241, purchased from Gibco).

[0096] The following steps were taken to conduct an immunization experiment in mice:

[0097] (1) Take out the lyophilized adjuvant powder stored in a 4℃ medical refrigerator, heat it in a 42℃ water bath for 15 min, and then inject 500 μL of PBS pH 7.2 basic (1×) using a 1 mL medical syringe. Take 1 mg of antigen protein, add PBS pH 7.2 basic (1×) to 500 μL and mix well, then mix with the above mixture and vortex thoroughly for 5 min. Aspirate with a 1 mL medical syringe to remove air bubbles from the syringe.

[0098] (2) For the first immunization of mice, 200 μL / mouse (200 μg antigen protein) was injected subcutaneously at two points, one in the peritoneum and one in the dorsal spine, with 50 μL injected at each point. The second immunization was performed 3 weeks after the first immunization, in the same manner as the first immunization. The third immunization was performed 3 weeks later in the same manner as the second immunization. One week later, tail vein blood was collected from mice to determine antibody titers. Mice with good titers were selected for a pulse immunization 3 days before fusion, i.e., immunization was performed by intraperitoneal injection of 100 μg of antigen protein diluted in 100 μL without adjuvant. The pulse immunization could be performed without adjuvant.

[0099] (3) Potency determination: 10 μL of mouse tail vein blood was dissolved in 490 μL of PBS, and serially diluted 3-fold from 1:3000 to 240,000. 100 μL of each solution was aspirated into ELISA plates coated with the target protein (100 ng / well) and incubated at 37°C for 1 h. The plates were washed 3 times with PBST washing buffer to remove residual solution. 100 μL of enzyme-labeled secondary antibody diluted with blocking buffer (1:10000) was added to each well and incubated at 37°C for 1 h. The plates were washed 4 times with PBST washing buffer to remove residual solution. 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) single-component chromogenic solution was added to each well and incubated in the dark for about 8 min. 50 μL of 2M H2SO4 stop solution was added to terminate the reaction, and the OD was read by a microplate reader. 450 The effective value was measured to be P / N > 2.1.

[0100] 1.3 Hybridoma Fusion

[0101] Materials and reagents

[0102] Hybridoma fusion experiment steps:

[0103] (1) SP2 / 0 cell preparation: Cells were revived in T75 cell culture flasks and passaged to T175 cell culture flasks on the third day. Subculture was performed daily thereafter, with a ratio of 1:2, 1:2.5, or 1:3 depending on the cell usage time and cell number. The culture medium used for SP2 / 0 cells was complete medium (DMEM medium containing 20% ​​FBS and 1% P / S).

[0104] (2) Spleen cell collection: Immunized mice were euthanized by cervical dislocation. The mice were immersed in 75% alcohol for 10 minutes for thorough disinfection. The mice were then fixed on the dissection table in a biosafety cabinet. The skin on the left side of the abdomen was lifted to expose the spleen. Surgical instruments were changed, the peritoneum was cut open, and surgical instruments were changed again. The spleen was carefully removed with curved forceps and placed in a 6-well plate containing 3 mL of empty DMEM culture medium. The plate was gently washed and the surrounding connective tissue was carefully removed.

[0105] Transfer the spleen to another petri dish containing 8 mL of empty DMEM culture. Use a 5 mL syringe to slowly expel the cells from the spleen using the empty DMEM. Then, pass the culture medium through a 70 μm mesh. Use a grinding stick to thoroughly grind the spleen on this mesh, ensuring the mesh is pre-wetted and kept moist until grinding is complete. Minimize cell loss; typically, one mouse yields 1 × 10⁶ cells. 8 ~2.5×10 8 One spleen cell.

[0106] (3) Cell electrofusion: At room temperature, centrifuge at 400g for 5 min, remove the supernatant, agitate the precipitated cells with 2 mL of erythrocyte lysis buffer, add another 13 mL of erythrocyte lysis buffer, let stand for 15 min, then centrifuge at 400g for 10 min, remove the supernatant, agitate the precipitated cells with 3 mL of empty culture DMEM, and take 8 × 10⁻⁶ cells. 7 ~9×10 7 Centrifuge SP2 / 0 myeloma cells at 200g for 5 min, wash twice with empty culture DMEM, then pass through a 70μm mesh sieve and mix with the above spleen cells. Centrifuge at 400g for 5 min with 15 mL of electrofusion medium (Cytofusion Medium C), wash twice with electrofusion medium, then blow the cells with 20 mL of medium to mix them, and begin electrofusion.

[0107] (4) Before electrofusion, rinse the electrode cup three times with Cytofusion Medium C, and measure the resistance during the last rinse. Add 4 mL of Cytofusion Medium C containing cells to the electrode, taking care to avoid air bubble formation, and press the Ω key to measure the resistance value. Click start to begin the electrofusion process. When three consecutive beeps are heard and the start key lights up again, the fusion is complete. Let stand for 2 minutes, then gently add the cells to a conical tube containing 40 mL of HAT selective medium (DMEM medium containing 20% ​​FBS, 1% HAT, 1% P / S, and 1% OPI) and incubate at 37°C for 60 minutes. Gently dilute the cells in the conical tube and plate them at 200 μL / well, and incubate at 37°C in a 5% CO2 incubator.

[0108] 1.4 Supernatant forward and reverse screening ELISA detection

[0109] Supernatant forward and reverse screening ELISA detection experimental steps:

[0110] 1. Coat with streptavidin (S9171, Solarbio) at a concentration of 100 ng / well and incubate overnight at 4°C;

[0111] 2. After pouring out the coating solution, wash twice with PBST, add 10 ng / well of N16-biotin (reverse screening group) or P16-biotin (positive screening group), incubate at 37℃ for 30 min, pour out the solution, and wash three times with PBST.

[0112] 3. Add 80 μL of cell supernatant to the detection plate and set up a positive control (mouse positive serum, diluted 1:1000).

[0113] Incubate at 4.37℃ in a biochemical incubator for 1 hour.

[0114] 5. Wash three times with PBST as washing solution and shake off any residual solution.

[0115] 6. Add 100 μL of enzyme-labeled secondary antibody (58-173-090418, Invitrogen) diluted with PBST + 2% BSA (1:10000) to each well.

[0116] 7. Incubate at 37℃ in a biochemical incubator for 1 hour.

[0117] 8. Wash four times with PBST as washing solution and pat off any residual solution.

[0118] 9. Add 100 μL of TMB single-component colorimetric solution (PR1200, Solarbio) to each well. After developing the color in the dark for about 8 minutes, add 50 μL of 2M H2SO4 to stop the reaction.

[0119] 10. Reading OD using an ELISA reader 450 Cell subcloning was performed using values ​​from the positive screening group and the negative screening group. Experimental results:

[0120] Four clones with P16 binding activity but no N16 binding activity were selected through forward and reverse screening.

[0121] 1.5 Cell Subcloning

[0122] Cell subcloning experimental procedures

[0123] Hybridoma cells were seeded evenly in each well using a limiting dilution method at a density of single cells per well, and positive wells containing single cell clusters were detected by Cell Base Elisa assay.

[0124] (1) Gently pipette the cells in the 12-well plate to mix them evenly. Use a pipette to draw 20 μL of cells and mix them with 20 μL of trypan blue. Count the cells using a cell counter (Countstar). Based on the counting results, transfer 300 cells to 40 ml of complete culture medium. The reagent composition of the complete culture medium is: 20% FBS + 1% P / S + 1% HAT + 1% OPI + DMEM.

[0125] (2) Spread 40ml of culture medium evenly on the plate, 200ml / well, spread on two plates, and place them in a 37℃ carbon dioxide incubator for incubation.

[0126] (3) On the 5th day, observe the cell state and the number of cell lines in each well under a microscope and mark them.

[0127] (4) After 7 days, positive wells were detected by indirect ELISA. The cell lines that were both single and positive were expanded to 24-well plates and cultured. Two days later, they were expanded to 6-well plates. When they grew to a density of 70%-80%, they were frozen. Two cells of each cell line were frozen and stored in liquid nitrogen.

[0128] The cloned hybridoma cells were sequenced and purified.

[0129] After domesticating the four clones, purified corresponding antibodies were obtained. Finally, four antibodies were selected from the four hybridomas and confirmed by sequencing for subsequent humanization.

[0130] Example 2: Humanization and Expression of Mouse Monoclonal Antibodies

[0131] 2.1 Humanization

[0132] The four antibodies obtained in Example 1 were humanized using NCBI in two ways: hot spot site (reverse mutation) and complete mutation of the hot spot site (non-reverse mutation), and then expressed. The experimental steps are as follows:

[0133] (1) Streak the preserved monoclonal antibody strain on a plate and incubate overnight at 37°C.

[0134] (2) Select single clones and activate them for 8 hours. Inoculate them into 250 ml of 2×YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) and shake overnight at 37°C.

[0135] (3) Plasmids extracted from bacterial culture (extracted using an endotoxin-free plasmid extraction kit (DP117, Tiangen Biotech)) were used for transfection expression in 293F cells, with a heavy chain to light chain vector ratio of 1:1.

[0136] (4) Plasmid transfection was performed according to the 293F cell transfection instructions (K70101, Zhuhai Kairui). Cell viability was measured daily after transfection. When the viability dropped to about 80%, the protein was collected, centrifuged at 1000 rpm for 10 min, and then centrifuged at 8000 rpm for 15 min. The supernatant was filtered through 0.8 μm and 0.45 μm filter membranes, respectively.

[0137] (5) Use a protein purification instrument to recover the protein from the protein A column.

[0138] (6) Centrifuge at 4000 rpm in a 30 kDa ultrafiltration tube at 4 °C. Replace the buffer with PBS three times. On the last time, transfer the antibody in the filter membrane to an EP tube and filter the antibody through a 0.22 μm filter membrane.

[0139] (7) The concentration of the antibody after filtration was determined using a BCA kit (P0010, Beyotime), and the remaining antibody was stored at -80℃.

[0140] After expression of the humanized antibodies, four active antibodies were obtained that can bind to the N3pE form of abeta, but bind weakly to the wild-type abeta. The specific humanized sequences are shown in Table 1 below.

[0141] Table 1 Humanization Sequences

[0142] Heavy chain constant region sequence:

[0143] Light chain constant region sequence:

[0144] 2.2 Determination of humanized antibodies

[0145] The four humanized antibodies BP21-2, P3-B31, BH7-4, Bh39A, mIgG2a, Lecanemab, and Donanemab obtained in 2.1 were subjected to antigen binding assays as described in Example 1 using a conventional ELISA experiment. Among them, mIgG2a was an isotype control, Lecanemab was an Aβ antibody that could bind to both wild-type and N3pE form of abeta, and Donanemab was a laboratory-prepared positive control antibody.

[0146] The binding activity profile is shown in Figure 1. As can be seen from Figure 1, the four humanized antibodies BP21-2, P3-B31, BH7-4, and Bh39A can bind to the N3pE form of abeta with strong activity, while their binding to the wild-type form of abeta is weaker or nonexistent. This characteristic differs from that of Lecanemab, which can bind to both wild-type and N3pE forms of abeta, but is similar to that of Donanemab.

[0147] Example 3: Antibody Affinity Assay

[0148] The experimental steps for detecting antibody affinity are as follows:

[0149] (1) Dilute 10×HBS-P+ buffer (BR100671, cytiva) 10 times to the working concentration (1×), filter it through a 0.22μm filter membrane, and remove air bubbles by sonication.

[0150] (2) Dilute the antibody to 1 μg / ml with diluted HBS-P+ buffer and determine the capture antibody level to determine the optimal antibody concentration.

[0151] (3) Dilute the antigen with diluted HBS-P+ buffer and conduct preliminary experiments to determine the maximum saturation concentration of the antigen.

[0152] (4) Dilute the antigen down to 7 concentrations based on the maximum saturation concentration, add 0 concentration to form 8 concentrations and the optimal antibody concentration for formal testing.

[0153] (5) Fit the curve based on the formal experimental results and calculate the KD, Kd and Ka values.

[0154] Experimental Results: The affinity of Bh39A and the control drug Donanemab for the polymeric N3pE form of abeta was obtained using surface plasmon resonance (SPR) assays, and the affinity data are shown in Table 2 below. As can be seen from Table 2, since the antigen is a polypeptide, the affinity is within 10 nM. Specifically, the KD of Bh39A is 19.2 nM, and the KD of Donanemab is 61.5 nM.

[0155] Table 2

[0156] Further verification and comparison were conducted on the binding of Bh39A antibody and Donanemab to monomeric and polymeric abeta, respectively. The results are shown in Figure 3. In the monomer binding assay, neither antibody bound to wild-type abeta (1-42) (Aβ antibody affinity KD > 1 μM can be considered as no binding), but both bound to N3pE abeta (p3-42). In the polymer binding assay, both antibodies bound very weakly to wild-type abeta (1-42) but very strongly to N3pE abeta (p3-42). The figure also shows that the Bh39A group bound significantly stronger to both monomeric and N3pE abeta (p3-42) than the Donanemab group.

[0157] The above experiments demonstrate that Bh39A (KD = 19.2 nM) not only has a stronger affinity than Donanemab (KD = 61.5 nM), but also has a stronger ability to bind the N3pE form of abeta (p3-42) than Donanenamab.

[0158] Example 4: Off-target flow cytometry cell binding assay

[0159] Experimental steps

[0160] (1) Prepare a PBS buffer containing 2% FBS. The PBS mentioned below refers to this buffer.

[0161] (2) Collect enough cells, centrifuge at 300g for 5 minutes, remove the supernatant, and resuspend in PBS.

[0162] (3) Spread the cells into a 96-well plate, 100 μL per well, with the number of cells per well required to be between 50,000 and 500,000.

[0163] (4) Centrifuge at 400g for 5 min at 4℃, discard the supernatant, resuspend the cells in 100μL of PBS diluted antibody, and incubate at 4℃ for 1-2 h.

[0164] (5) Centrifuge at 400g, 4℃ for 5min, discard the supernatant, resuspend in 200μL PBS, centrifuge to remove the supernatant, and repeat once.

[0165] (6) Resuspend cells in 100 μL of fluorescent secondary antibody (A0556, Beyotime) diluted in PBS and incubate at 4°C for 1-2 h.

[0166] (7) Centrifuge at 400g, 4℃ for 5min, discard the supernatant, resuspend in 200μL PBS, centrifuge again to remove the supernatant, and repeat once. Finally, resuspend the cells in 100μL PBS, detect fluorescence by flow cytometry, compare with blank control, and use 14F3 and 24B6 antibodies from WO2024141099A1 for negative and positive controls, respectively, to determine the off-target effect of humanized antibodies on cells.

[0167] Off-target assay results are shown in Table 3 below. -50 / -200 represents the concentration (μg / mL) of the corresponding antibody in the resuspended cell sample, and the percentage represents the positive proportion of target cells that can bind the corresponding antibody. As can be seen from the table, no off-target binding was detected with the four antibodies Bh39A, BH7-4, P3B31, and BP21-2 to these target cell lines.

[0168] Table 3

[0169] Example 5: Determination of Aβ protein levels in APP / PS1 mice

[0170] Materials and reagents:

[0171] Six-month-old female APP / PS1 mice were purchased from Cyagen Biosciences;

[0172] 21F12 and 3D6-HRP are self-made antibodies (prepared with reference to patents WO2014007982 and WO2002046237);

[0173] Brain tissue lysis buffer: 5.5M guanidine / 50mM Tris / 0.5× protease inhibitor mixture (pH=8.0);

[0174] Standard sample: aβ1-42 (Qiangyao Biotechnology), dissolved in 10mM NaOH, final concentration 0.5mg / mL;

[0175] Single-component TMB colorimetric solution, Solarbio, PR1200-1000ml;

[0176] ELISA stop solution, Solarbio, C1058-500ml;

[0177] Dosing regimen and brain tissue treatment in APP / PS1 mice:

[0178] Dosage regimen: 6-month-old female APP / PS1 mice were administered the drug at a dose of 15 mg / kg / week for a total of 12 weeks;

[0179] Four antibodies, along with the positive control antibodies Donaneamb, PBS, and mIgG2a, were injected into APP / PS1 model mice to detect the effects of the above-mentioned drug administration on APP / PS1 mice. Specifically, 6-month-old APP / PS1 mice were injected weekly with 15 mg / kg of the drug or PBS for 12 consecutive weeks. On days 6-7 after the last administration, the right hemisphere of the mouse brain was harvested, homogenized, and the tissue homogenate was used. Organic solvents such as guanidine hydrochloride were used to dissolve the abeta aggregates in the plaques, and the total abeta level in the right hemisphere was detected using ELISA.

[0180] Mouse brain tissue processing: The mouse brain tissue was completely removed, and the surface blood was rinsed with pre-cooled physiological saline. The left hemisphere was separated on ice and fixed in 4% paraformaldehyde at 10 times the volume of the brain for 24 hours. The material was used for frozen section staining. The right hemisphere was weighed and frozen at -80°C for subsequent lysis in this experiment.

[0181] Preparation of brain tissue lysis buffer: 5.25 g guanidine hydrochloride + 0.06057 g Tris + water to a final volume of 10 ml, adjust pH to 8.0 with concentrated hydrochloric acid, and add 50 μL of a protease inhibitor mixture (Beyotime, P1020). Brain tissue sample dilution buffer was obtained by diluting brain tissue lysis buffer (5.5 M guanidine hydrochloride concentration) with 2% BSA / PBST.

[0182] Experimental steps for measuring Aβ protein levels:

[0183] (1) Coating: 21F12 was diluted with CBS to 15 μg / mL, 100 μL per well, and incubated overnight at 4°C.

[0184] (2) Blocking: Discard the coating solution, add 200 μL of 2% BSA / PBST to each well, and incubate at 37°C for 2 h.

[0185] (3) Blot off, wash once with 200μL / well PBST.

[0186] (4) Sample addition:

[0187] a. Sample dilution:

[0188] b. Standard product dilution:

[0189] aβ 1-42 First, dilute with brain tissue lysis buffer to 4 μg / ml (4 μL aβ). 1-42 +496μL brain tissue lysis fluid).

[0190] Standard dilution for well 1: 25 μL 4 μg / ml standard + 225 μL 2% BSA / PBST (concentration 400 ng / ml); for the remaining 11 wells, first add 120 μL 0.55M brain tissue lysis buffer (2% BSA / PBST diluted 10 times), then add 120 μL + 120 μL 2X serial dilution to well 11, and do not add standard to the last well.

[0191] c. Add 100 μL / well of the diluted sample and standard to the sealed ELISA plate and incubate at 37°C for 1 h.

[0192] (5) Remove the water, wash 4 times with 200 μL / well PBST, add 100 μL 3D6-HRP (diluted with 2% BSA / PBST to 1000X) to each well, incubate at 37℃ for 1 h.

[0193] (6) Tap off, wash 4 times with 200μL PBST.

[0194] (7) Add 100 μL / well TMB for color development, add 50 μL / well ELISA stop solution to terminate the reaction, and read the OD450nm value on the infinite F50 instrument.

[0195] Experimental results: The abeta level data obtained are shown in Figure 2. As can be seen from Figure 2, after 12 weeks of drug administration, in the mice of each group, the positive control group Donanemab showed only a slight decrease in abeta level, and there was no significant difference compared with the PBS group; while Bh39A showed a more significant decrease in abeta level compared with the PBS group, and there was a significant difference (p = 0.0106).

[0196] Example 6: Histochemical detection of APP / PS1 mice

[0197] Left hemisphere sections (maximum cross-section) were taken from uniform locations in both the Bh39A and Donanemab groups for histochemical staining to assess plaque formation. Sections were stained with 3D6 (abeta antibody, targeting plaques) and nuclear solid red, then observed and photographed under a microscope, with five different fixed locations photographed for each section. Images were analyzed using ImageJ, and the Area % (percentage of positive area) and Intensity Density (density of positive area) were statistically analyzed.

[0198] To assess the risk of brain microbleeds from Bh39A in APP / PS1 mice, we stained sections with Prussian blue, a staining method that indicates the location and size of microbleeds in brain tissue.

[0199] Plaque detection experimental steps

[0200] (1) Material processing: Take out frozen sections of brain tissue from -80℃ and dry them at 4℃ for about 15 minutes. Rinse with PBS for 5 minutes, 3 times.

[0201] (2) Blocking endogenous peroxidase: Discard the PBS solution, draw a circle around the tissue with a PAP pen, add endogenous peroxidase blocking solution (PO100A, Beyotime), and incubate at room temperature for 10 min. Rinse with PBS for 5 min, 3 times. Set aside.

[0202] (3) Cell permeability: Cell membrane permeability was increased by punching holes with immunostaining permeability solution (Triton X-100) (P0096, Beyotime), and the sections were incubated at room temperature for 10 min.

[0203] (4) Blocking non-specific binding sites: Block with immunostaining blocking solution (P0260, Beyotime), and incubate the sections at room temperature for 30 min.

[0204] (5) Antibody incubation: Gently shake dry and wipe the slide and surrounding area with filter paper to remove excess serum blocking solution. Add HRP-labeled 3D6 antibody dilution solution without rinsing. Set up a negative control by adding 0.01M PBS to the control tissue. Then place the slide in a humidifier and incubate overnight at 4°C to reduce background staining.

[0205] (6) Warming: After incubating the antibody overnight at 4°C, warm it to room temperature for about 45 minutes. This is to prevent the slide from detaching if it is placed directly into PBS for rinsing at 4°C, and to make the antigen-antibody binding more stable. Rinse with PBS for 5 minutes, 3 times.

[0206] (7) DAB color development: Add freshly prepared diaminobenzidine (DAB) working solution (34065, Thermo). Control the color development time under a microscope for 5 minutes. After the color development is complete, place the solution in tap water to stop the color development.

[0207] Prussian blue staining experimental steps (1) Counterstaining with Prussian blue: After DAB staining is completed, rinse with PBS for 5 min, 3 times. Add freshly prepared Perls staining working solution (G1422, Solarbio) and cover the section for staining for 30 min. Rinse with distilled water for 10 min.

[0208] (3) Cell nuclear staining: stain with nuclear solid red staining solution for 15 seconds, then stop the staining by placing the solution in tap water.

[0209] Sealing and observation

[0210] (1) Dehydration: 70%, 80%, and 90% ethanol for 3-5 seconds each, and 100% ethanol twice for 1 minute each time.

[0211] (2) Clearing: Clear the tissue twice with an environmentally friendly tissue clearing and dewaxing solution, each time for 1-2 minutes. Mount the slide with neutral resin, let it dry, and then observe it under a microscope.

[0212] Data processing

[0213] (1) Aβ plaque detection

[0214] Under a microscope, brown spots on the DAB staining result indicate positive Aβ plaques. Images were taken from the five fields of view with the most plaques under a 10x objective lens. The area percentage of each group of plaques was subsequently calculated.

[0215] (2) Microbleed detection

[0216] Under a microscope, hemosiderin or ferric iron is stained blue, and the cell nucleus is stained red.

[0217] (3) Statistical analysis of Aβ plaques

[0218] Select all positive areas and calculate the percentage of patch area.

[0219] Experimental results: The percentage of positive area and the statistical results of positive area intensity density are shown in Figure 4. The staining images of sections with the median percentage of positive area in the three groups of treated model mice are shown in Figure 5.

[0220] As can be seen, the Bh39A group was superior to the PBS group in both the percentage of positive area (p = 0.0011) and the intensity density of positive areas (p = 0.0008). In contrast, there was no significant difference between the Donanemab group and the PBS group (percentage of positive area: p = 0.2974, intensity density of positive areas: p = 0.2715). Therefore, in the APP / PS1 model mice, Bh39A has a greater advantage over Donanemab in clearing plaques in the mouse brain.

[0221] The blue spots in the Prussian blue stained sections were statistically analyzed for large, medium, and small (typical images of spots in each group and illustrations of large, medium, and small spots are shown in Figure 6). The statistical results are shown in Table 4 below.

[0222] Table 4

[0223] As can be seen from Figure 6 and Table 4 above, compared with the PBS group, the Bh39A group did not show more microbleeds, while the Donanemab group showed significantly more microbleeds than the PBS group.

[0224] Example 7: Bh39A rhesus monkey pre-PK experiment

[0225] A male rhesus monkey was given a single subcutaneous injection of 10 mg / kg of Bh39A. Plasma samples were collected over 28 days (specific sampling times were 0 h before administration, 5 h after administration, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 16 days, 20 days, 24 days, and 28 days after administration). Blood drug concentration was detected using ELISA, and PK data were analyzed using DAS2.0 with a two-compartment model. As shown in Table 5 below, the half-life was 200.4 h.

[0226] Table 5

[0227] Conclusion: Bh39A has a stronger ability to bind to the target N3pE form of abeta than Donaneamb: Bh39A exhibits high specificity for binding to abeta (p3-42) but not to wild-type abeta, and according to ELISA and SPR results, Bh39A has a stronger ability to bind to the N3pE form of abeta than positive Donaneamb.

[0228] Bh39A demonstrated superior abeta plaque clearance compared to Donanemab in APP / PS1 mice: APP / PS1 mice receiving Bh39A showed significantly lower abeta levels in brain tissue compared to the PBS group (p = 0.0006), while the Donanemab group showed no significant difference. Furthermore, in brain tissue staining (3D6-HRP), Bh39A treatment was superior to the PBS group in both the percentage of positive area (p = 0.0011) and the intensity density of positive areas (p = 0.0008). In contrast, there was no significant difference between the Donanemab and PBS groups (percentage of positive area: p = 0.2974, intensity density of positive areas: p = 0.2715).

[0229] APP / PS1 model mice treated with Bh39A did not show more cerebral microbleeds: compared with the PBS group, the Bh39A group did not show more microbleeds, while the Donanemab group showed significantly more microbleeds than the PBS group.

[0230] In summary, the humanized antibody Bh39A exhibits stronger specific binding to abeta (p3-42) and better in vivo plaque clearance than the control drug Donanemab, without showing more cerebral microbleeds.

Claims

1. An antibody or antigen-binding fragment thereof that binds to amyloid β (Aβ), comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3: The amino acid sequence of HCDR1 is selected from SEQ ID NO:1, 9, 17 and 25; The amino acid sequence of HCDR2 is selected from SEQ ID NO:2, 10, 18 and 26; The amino acid sequence of HCDR3 is selected from SEQ ID NO:3, 11, 19 and 27; The amino acid sequence of LCDR1 is selected from SEQ ID NO:4, 12, 20 and 28; The amino acid sequence of LCDR2 is selected from SEQ ID NO:5, 13, 21 and 29; and / or The amino acid sequence of LCDR3 is selected from SEQ ID NO:6, 14, 22 and 30.

2. The antibody or its antigen-binding fragment according to claim 1, The amino acid sequence of its heavy chain variable region is selected from SEQ ID NO:7, 15, 23 and 31; and / or The amino acid sequences of its light chain variable region are selected from SEQ ID NO:8, 16, 24 and 32.

3. An antibody or antigen-binding fragment thereof that binds to Αβ, wherein, The antibody or its antigen-binding fragment comprises any one of the following heavy chains HCDR1, HCDR2, and HCDR3 and light chains LCDR1, LCDR2, and LCDR3: (1) HCDR1 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, HCDR3 contains the amino acid sequence shown in SEQ ID NO:3, LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

6. (2) HCDR1 contains the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains the amino acid sequence shown in SEQ ID NO:10, HCDR3 contains the amino acid sequence shown in SEQ ID NO:11, LCDR1 contains the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

14. (3) HCDR1 contains the amino acid sequence shown in SEQ ID NO:17, HCDR2 contains the amino acid sequence shown in SEQ ID NO:18, HCDR3 contains the amino acid sequence shown in SEQ ID NO:19, LCDR1 contains the amino acid sequence shown in SEQ ID NO:20, LCDR2 contains the amino acid sequence shown in SEQ ID NO:21, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

22. (4) HCDR1 contains the amino acid sequence shown in SEQ ID NO:25, HCDR2 contains the amino acid sequence shown in SEQ ID NO:26, HCDR3 contains the amino acid sequence shown in SEQ ID NO:27, LCDR1 contains the amino acid sequence shown in SEQ ID NO:28, LCDR2 contains the amino acid sequence shown in SEQ ID NO:29, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

30.

4. An antibody or antigen-binding fragment thereof that binds to Αβ, wherein, The antibody or its antigen-binding fragment comprises HCDR of the heavy chain and LCDR of the light chain, as shown in any of the following groups: (1) HCDR1 of the amino acid sequence shown in SEQ ID NO:1, HCDR2 of the amino acid sequence shown in SEQ ID NO:2, HCDR3 of the amino acid sequence shown in SEQ ID NO:3, LCDR1 of the amino acid sequence shown in SEQ ID NO:4, LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

6. (2) HCDR1 of the amino acid sequence shown in SEQ ID NO:9, HCDR2 of the amino acid sequence shown in SEQ ID NO:10, HCDR3 of the amino acid sequence shown in SEQ ID NO:11, LCDR1 of the amino acid sequence shown in SEQ ID NO:12, LCDR2 of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

14. (3) HCDR1 of the amino acid sequence shown in SEQ ID NO:17, HCDR2 of the amino acid sequence shown in SEQ ID NO:18, HCDR3 of the amino acid sequence shown in SEQ ID NO:19, LCDR1 of the amino acid sequence shown in SEQ ID NO:20, LCDR2 of the amino acid sequence shown in SEQ ID NO:21, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

22. (4) HCDR1 of the amino acid sequence shown in SEQ ID NO:25, HCDR2 of the amino acid sequence shown in SEQ ID NO:26, HCDR3 of the amino acid sequence shown in SEQ ID NO:27, LCDR1 of the amino acid sequence shown in SEQ ID NO:28, LCDR2 of the amino acid sequence shown in SEQ ID NO:29, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

30.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein, The antibody or its antigen-binding fragment binds to Aβ in the form of N3pE; preferably, the antibody or its antigen-binding fragment does not bind to monomeric wild-type Aβ, and / or, the antibody or its antigen-binding fragment substantially does not bind to polymeric wild-type Aβ.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein, The antibody has any one of the following sets of heavy chain variable region sequences and light chain variable region sequences: (1) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:8; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:8; (2) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:15, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:16; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:15 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:16; (3) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:23, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:24; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:23 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:24; (4) A heavy chain variable region having 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 the amino acid sequence shown in SEQ ID NO:31, and a light chain variable region having 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 the amino acid sequence shown in SEQ ID NO:32; preferably, the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:31 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:

32.

7. The antibody or antigen-binding fragment thereof as described in any one of claims 1-6, wherein, The antibody also has one or more of the following characteristics: (1) It further includes a heavy chain constant region and / or a light chain constant region; (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; (3) It is a monoclonal antibody; (4) It is a full-length antibody, or it is a Fab, Fv, scFv, F(ab')2, linear antibody or single-domain antibody; (5) It is in the form of IgG1, IgG2, IgG3 or IgG4; (6) It binds to the form of polymer N3pE with an affinity of less than 20 nM.

8. The antibody or antigen-binding fragment according to claim 7, wherein the heavy chain constant region comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:33; and the light chain constant region comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:34; Preferably, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:33; the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:34; Preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:33; and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:

34.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein each CDR region is defined according to the Kabat definition scheme, the Chothia definition scheme, the Abm definition scheme, the IMGT definition scheme, and / or the Contact definition scheme.

10. A fusion protein, wherein one fused portion comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

11. A bispecific antibody or a multispecific antibody, wherein one antigen-binding domain comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-9.

12. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-9.

13. An expression vector comprising the nucleic acid molecule according to claim 12.

14. Transformation or transfection of host cells with the expression vector according to claim 13.

15. A method for preparing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell of claim 14 under conditions suitable for the production of said antibody or antigen-binding fragment thereof; and isolating said antibody or antigen-binding fragment thereof from said host cell and / or culture.

16. An antibody-drug conjugate comprising an antibody or antigen-binding portion thereof as described in any one of claims 1-9, or a bispecific or multispecific antibody as described in claim 11, covalently attached to a therapeutic portion; preferably, the therapeutic portion is selected from cytotoxic drugs, cytokines, immunosuppressants, immunostimulants, cleaved peptides, and radioisotopes.

17. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-9, a fusion protein according to claim 10, a bispecific antibody or multispecific antibody according to claim 11, a nucleic acid molecule according to claim 12, an expression vector according to claim 13, a host cell according to claim 14 or an antibody-drug conjugate according to claim 16, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

18. An antibody or antigen-binding fragment thereof according to any one of claims 1-9, a fusion protein according to claim 10, a bispecific antibody or multispecific antibody according to claim 11, a nucleic acid molecule according to claim 12, an expression vector according to claim 13, a host cell according to claim 14, an antibody-drug conjugate according to claim 16, or a pharmaceutical composition according to claim 17, for the diagnosis or treatment of diseases associated with Aβ aggregation and / or deposition in the brain of a subject; Preferably, the diseases associated with Aβ accumulation and / or deposition in the subject's brain include amyloidosis and neurological diseases; more preferably, the amyloidosis includes secondary amyloidosis and age-related amyloidosis; and / or, the neurological disease is Alzheimer's disease, more preferably Alzheimer's disease-related mild cognitive impairment, mild Alzheimer's disease-related dementia, and familial early-onset Alzheimer's disease.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-9, the fusion protein according to claim 10, the bispecific antibody or multispecific antibody according to claim 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, the host cell according to claim 14, the antibody-drug conjugate according to claim 16, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for the diagnosis or treatment of diseases associated with Aβ aggregation and / or deposition in the brain of a subject; Preferably, the diseases associated with Aβ aggregation and / or deposition include amyloidosis and neurological diseases; more preferably, the amyloidosis includes secondary amyloidosis and age-related amyloidosis; and / or, the neurological disease is Alzheimer's disease, more preferably Alzheimer's disease-related mild cognitive impairment, mild Alzheimer's disease-related dementia, and familial early-onset Alzheimer's disease.

20. A method for diagnosing or treating a disease associated with Aβ accumulation and / or deposition in the brain of a subject, comprising administering to a subject in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, a fusion protein as described in claim 10, a bispecific or multispecific antibody as described in claim 11, a nucleic acid molecule as described in claim 12, an expression vector as described in claim 13, a host cell as described in claim 14, an antibody-drug conjugate as described in claim 16, or a pharmaceutical composition as described in claim 17; Preferably, the diseases associated with Aβ accumulation and / or deposition in the subject's brain include amyloidosis and neurological diseases; more preferably, the amyloidosis includes secondary amyloidosis and age-related amyloidosis; and / or, the neurological disease is Alzheimer's disease, more preferably Alzheimer's disease-related mild cognitive impairment, mild Alzheimer's disease-related dementia, and familial early-onset Alzheimer's disease.

21. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-9, a fusion protein according to claim 10, a bispecific antibody or multispecific antibody according to claim 11, a nucleic acid molecule according to claim 12, an expression vector according to claim 13, a host cell according to claim 14, an antibody-drug conjugate according to claim 16, or a pharmaceutical composition according to claim 17.

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