A [beta] protofibril antibody or functional fragment thereof and application thereof

Abstract

The invention relates to the field of antibody treatment, in particular to an A beta primary fibril antibody or a functional fragment thereof and application thereof. According to the present invention, the existing Lancanamazumab is modified, histidine mutation is introduced into the CDR region of the antibody, and the mutant having high affinity with the antigen under the high pH condition and having low affinity or no affinity with the antigen under the low pH condition is screened, such that the combination of the modified antibody and the antigen presents the pH dependent form, such that the pH dependent form is provided; in some embodiments, the mutant reduces binding to the antigen in a low pH environment of the lysosome (about pH 4.5 to about pH 5.0), such that when the antigen degrades in the lysosome, the antibody can be recycled back into the blood, thereby increasing the recycling of the antibody.

Description

Technical Field

[0001] The present invention relates to the field of antibody therapy, and in particular to Aβ protofibril antibodies or functional fragments thereof and applications thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease whose hallmarks include extracellular amyloid plaques, intraneuronal neurofibrillary tangles, neuronal dysfunction and eventual brain atrophy. AD symptoms include memory impairment, aphasia, apraxia, agnosia and visual-spatial impairment, and the dementia caused by it accounts for 60% to 80% of all dementia cases. The risk of AD increases with age, seriously affecting the quality of life of the elderly and causing great distress to families and society.

[0003] Current research suggests that the aggregation and excessive deposition of amyloid β-protein (Aβ) triggers a series of cascade reactions such as tau protein hyperphosphorylation and neuronal damage, which are the main causes of Alzheimer's disease. Lecanemab, an antibody that specifically targets Aβ, was approved for accelerated approval by the FDA in January 2023, becoming the second innovative Alzheimer's disease therapy after aducanumab. On January 9, 2024, Lecanemab injection (trade name: Leqembi) was also approved for marketing in China, with indications for the treatment of mild cognitive impairment caused by Alzheimer's disease and mild dementia caused by Alzheimer's disease.

[0004] Lencanicarboreumab is a humanized anti-Aβ antibody that binds to repetitive conformational epitopes in the protofibril form of amyloid-β, promoting the clearance of abnormally accumulated amyloid-β in the brains of patients with Alzheimer's disease, thereby changing the disease pathology and alleviating the progression of the disease. Studies have shown that when treated with Lencanicarboreumab for 6 months, there was a significant statistical difference between the experimental group and the placebo group. After 18 months of treatment, the rate of decline in cognitive and memory function in the experimental group slowed by 27%. In addition, Lencanicarboreumab can also significantly reduce the amyloid load in the patient's brain and delay tau deposition. According to model predictions, Lencanicarboreumab delays the progression of early Alzheimer's patients to moderate disease by 2-3 years.

[0005] Due to the special nature of AD degenerative diseases, lencanezumab needs to be administered by long-term injection at a frequency of once every two weeks. Therefore, improving the antibody's in vivo half-life and / or reducing its in vivo clearance rate are of great value in increasing the convenience and compliance of the drug and reducing the cost of medication. Summary of the invention

[0006] The present invention modifies lencanezumab by introducing at least one histidine mutation into the antibody so that the binding of the antibody to the antigen is pH-dependent, and a mutant is obtained whose affinity to the antigen is significantly reduced under low pH conditions but whose affinity remains substantially unchanged under physiological pH conditions. Thus, in the low pH environment of the lysosome (pH 4.5-5), the antibody of the present invention can dissociate from the antigen with an increased frequency and recycle back into the cell, thereby reducing the antigen-mediated antibody clearance effect and maximizing the average number of times that the unit antibody binds to the antigen.

[0007] One object of the present invention is to provide an Aβ protofibril antibody or a functional fragment thereof, which has at least one histidine mutation relative to the amino acid sequence of lencanezumab and comprises an antibody heavy chain having an amino acid sequence as shown in any one of SEQ ID NO: 2, 3, 4, 6 or 7, or a combination of VH-CDR1, VH-CDR2 and VH-CDR3 in the variable region of the antibody heavy chain having an amino acid sequence as shown in any one of SEQ ID NO: 19, 20, 22, 26 or 28, and / or an antibody light chain having an amino acid sequence as shown in SEQ ID NO: 9 or 10, or a combination of VL-CDR1, VL-CDR2 and VL-CDR3 in the variable region of the antibody light chain having an amino acid sequence as shown in SEQ ID NO: 30 or 32.

[0008] In some preferred embodiments, the Aβ protofibril antibody or its functional fragment disclosed herein comprises a CDR combination selected from any one of (a) to (q): (a) a VH-CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as shown in SEQ ID NO: 21, a VH-CDR3 having an amino acid sequence as shown in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as shown in SEQ ID NO: 16, a VL-CDR2 having an amino acid sequence as shown in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as shown in SEQ ID NO: 18; (b) a VH-CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as shown in SEQ ID NO: 21, a VH-CDR3 having an amino acid sequence as shown in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as shown in SEQ ID NO: 16, a VL-CDR2 having an amino acid sequence as shown in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as shown in SEQ ID NO: 18. NO:17, and a VL-CDR2 having the amino acid sequence of SEQ ID NO:18; (c) a VH-CDR1 having the amino acid sequence of SEQ ID NO:12, a VH-CDR2 having the amino acid sequence of SEQ ID NO:23, a VH-CDR3 having the amino acid sequence of SEQ ID NO:14, a VL-CDR1 having the amino acid sequence of SEQ ID NO:16, a VL-CDR2 having the amino acid sequence of SEQ ID NO:17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO:18; (d) a VH-CDR1 having the amino acid sequence of SEQ ID NO:12, a VH-CDR2 having the amino acid sequence of SEQ ID NO:13, a VH-CDR3 having the amino acid sequence of SEQ ID NO:27, a VL-CDR1 having the amino acid sequence of SEQ ID NO:16, a VL-CDR2 having the amino acid sequence of SEQ ID NO:17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO:18. a VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and a VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18;(e) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 29, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; (f) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; (g) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18 NO:12, a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO:13, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO:14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO:33, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO:18; (h) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO:12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO:21, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO:14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO:31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO:18; (i) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO:12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO:21, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO:14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO:31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO:18 NO:12, a VH-CDR1 having the amino acid sequence set forth in SEQ ID NO:23, a VH-CDR2 having the amino acid sequence set forth in SEQ ID NO:14, a VL-CDR1 having the amino acid sequence set forth in SEQ ID NO:31, a VL-CDR2 having the amino acid sequence set forth in SEQ ID NO:17, and a VL-CDR3 having the amino acid sequence set forth in SEQ ID NO:18;(j) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 29, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; (k) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; (l) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: NO:12, a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO:13, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO:13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO:27, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO:31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO:18; (m) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO:12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO:13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO:14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO:33, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO:18; (n) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO:12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO:13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO:14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO:33, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO:18 NO:12, a VH-CDR1 having the amino acid sequence set forth in SEQ ID NO:21, a VH-CDR2 having the amino acid sequence set forth in SEQ ID NO:14, a VL-CDR1 having the amino acid sequence set forth in SEQ ID NO:33, a VL-CDR2 having the amino acid sequence set forth in SEQ ID NO:17, and a VL-CDR3 having the amino acid sequence set forth in SEQ ID NO:18;(o) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 23, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 33, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; (p) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 33, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; (q) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 33, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: NO:12, a VH-CDR1 having an amino acid sequence as shown in SEQ ID NO:29, a VH-CDR2 having an amino acid sequence as shown in SEQ ID NO:14, a VL-CDR1 having an amino acid sequence as shown in SEQ ID NO:33, a VL-CDR2 having an amino acid sequence as shown in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as shown in SEQ ID NO:18.;

[0009] In a more preferred embodiment, the antibodies or functional fragments thereof disclosed herein comprise an antibody heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 2, 3, 4, 6 or 7, or an antibody heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 19, 20, 22, 26 or 28, and / or an antibody light chain having an amino acid sequence as shown in SEQ ID NOs: 9 or 10, or an antibody light chain variable region having an amino acid sequence as shown in SEQ ID NOs: 30 or 32.

[0010] In some embodiments, the antibody further comprises a constant region. Optionally, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Optionally, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans; preferably, the constant region is derived from humans.

[0011] Optionally, the constant region is an IgG constant region. Optionally, the constant region is selected from the constant region of IgG1 or IgG4.

[0012] For example, in some embodiments, the sequence of the light chain constant region of the constant region is as shown in SEQ ID NO:35; in some embodiments, the sequence of the light chain constant region of the constant region is at least 80% identical to SEQ ID NO:35; in some embodiments, the sequence of the light chain constant region of the constant region is at least 85% identical to SEQ ID NO:35; in some embodiments, the sequence of the light chain constant region of the constant region is at least 90% identical to SEQ ID NO:35; in some embodiments, the sequence of the light chain constant region of the constant region is at least 92% identical to SEQ ID NO:35; in some embodiments, the sequence of the light chain constant region of the constant region is at least 95% identical to SEQ ID NO:35; in some embodiments, the sequence of the light chain constant region of the constant region is at least 98% identical to SEQ ID NO:35; in some embodiments, the sequence of the light chain constant region of the constant region is at least 99% identical to SEQ ID NO:35. In some embodiments, the light chain constant region of the constant region has an amino acid sequence with one or more positions substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO:35. In some embodiments, the light chain constant region of the constant region has one, two, three, four, five, six, seven, eight, nine or ten sites of substitution, deletion or insertion of an amino acid sequence compared to the amino acid sequence shown in SEQ ID NO:35.

[0013] For example, in some embodiments, the heavy chain constant region sequence of the constant region is as shown in SEQ ID NO: 34; in some embodiments, the heavy chain constant region sequence of the constant region has at least 80% identity with SEQ ID NO: 34; in some embodiments, the heavy chain constant region sequence of the constant region has at least 85% identity with SEQ ID NO: 34; in some embodiments, the heavy chain constant region sequence of the constant region has at least 90% identity with SEQ ID NO: 34; in some embodiments, the heavy chain constant region sequence of the constant region has at least 92% identity with SEQ ID NO: 34; in some embodiments, the heavy chain constant region sequence of the constant region has at least 95% identity with SEQ ID NO: 34; in some embodiments, the heavy chain constant region sequence of the constant region has at least 98% identity with SEQ ID NO: 34; in some embodiments, the heavy chain constant region sequence of the constant region has at least 99% identity with SEQ ID NO: 34. In some embodiments, the heavy chain constant region of the constant region has an amino acid sequence having one or more positions substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO: 34. In some embodiments, the heavy chain constant region of the constant region has an amino acid sequence with one, two, three, four, five, six, seven, eight, nine or ten sites substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO:34.

[0014] In some embodiments, the functional fragment of the Aβ protofibril antibody disclosed herein is selected from any one of Fab, Fab', F(ab')2, Fd, Fv, scFv and dAb.

[0015] In some embodiments, the Aβ protofibril antibody or its functional fragment disclosed herein can also be connected to other elements suitable for connection to the antibody or antibody fragment, or modified to be suitable for the intended use of the antibody or antibody fragment, including but not limited to elements or modifications that increase protein stability, elements or modifications that extend half-life, separation or purification tags, immune activation elements and / or in vivo delivery elements. In some embodiments, the in vivo delivery element is selected from a membrane-penetrating peptide, a nuclear localization signal, an organelle localization signal, an endoplasmic reticulum retention signal, a peroxisome targeting signal and / or a mitochondrial transit peptide. In some embodiments, the separation or purification tag includes an MBP tag and a His tag.

[0016] The second object of the present invention is to provide a nucleic acid molecule encoding the Aβ protofibril antibody or its functional fragment disclosed herein. In some embodiments, the nucleic acid molecule is a DNA molecule. In other embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is separated from its natural environment (such as the host cell environment). In some embodiments, the nucleic acid molecules disclosed herein encode a heavy chain variable region having a nucleotide sequence as shown in any one of SEQ ID NO: 38, 39, 40, 42 or 43, and / or a light chain variable region having a nucleotide sequence as shown in SEQ ID NO: 44 or 45. In some embodiments, the nucleic acid molecules disclosed herein can be codon optimized, preferably human codon optimized.

[0017] A third object of the present invention is to provide an expression vector comprising a nucleic acid molecule disclosed herein. In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector or a shuttle vector. In a preferred embodiment, the expression vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector.

[0018] The fourth object of the present invention is to provide a recombinant virus particle comprising the expression vector disclosed herein. In some embodiments, the recombinant virus particle is selected from a recombinant lentiviral particle, a recombinant adenoviral particle, a recombinant adeno-associated virus particle or a recombinant retroviral particle.

[0019] The fifth object of the present invention is to provide a host cell, which expresses the Aβ protofibril antibody or its functional fragment disclosed herein, or contains the nucleic acid molecule disclosed herein or the expression vector disclosed herein, or expresses the recombinant virus particle disclosed herein.

[0020] The sixth object of the present invention is to provide a composition comprising the Aβ protofibril antibody disclosed herein or its functional fragment, the nucleic acid molecule disclosed herein, the expression vector disclosed herein, the recombinant virus particle disclosed herein or the host cell disclosed herein. In some embodiments, the composition is a pharmaceutical composition, which also includes a pharmaceutically acceptable excipient.

[0021] The Aβ protofibril antibody or its functional fragment, the nucleic acid molecule disclosed herein, the expression vector disclosed herein, the recombinant virus particle disclosed herein, or the host cell disclosed herein provided by the present invention has the effect of clearing or reducing beta amyloid protein aggregates. Therefore, the seventh object of the present invention is to provide the use of the Aβ protofibril antibody or its functional fragment, the nucleic acid molecule disclosed herein, the expression vector disclosed herein, the recombinant virus particle disclosed herein, the host cell disclosed herein, or the composition disclosed herein in the preparation of a drug for treating or preventing a disease caused by beta amyloid protein aggregation or deposition.

[0022] The eighth object of the present invention is to provide a method for treating or preventing diseases caused by aggregation or deposition of β-amyloid protein, comprising administering to a subject in need thereof a therapeutically effective amount of the Aβ protofibril antibody or its functional fragment disclosed herein, the nucleic acid molecule disclosed herein, the expression vector disclosed herein, the recombinant virus particle disclosed herein, the host cell disclosed herein, or the composition disclosed herein.

[0023] In some embodiments, the disease caused by the aggregation or deposition of amyloid beta is a neurological disease. In some embodiments, the disease caused by the aggregation or deposition of amyloid beta includes traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tau disease, systemic amyloidosis, atherosclerosis, Parkinson's disease dementia (PDD) and Alzheimer's disease (AD). In some embodiments, the disease caused by the aggregation or deposition of amyloid beta is Alzheimer's disease. In some embodiments, the disease caused by the aggregation or deposition of amyloid beta is mild cognitive impairment and Alzheimer's disease mild dementia caused by Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The binding force determination curves of exemplary heavy chain mutants H_T28E, H_S53H, H_S56H, H_Y50H, H_Y109H and parent antibody Lecanemab of the present invention to Aβ protofibrils are shown;

[0025] Figure 2 The binding force determination curves of exemplary light chain mutants L_Q27H, L_S28H and parent antibody Lecanemab of the present invention to Aβ protofibrils are shown;

[0026] Figure 3 The binding force determination curves of exemplary mutant antibodies H_S53H / L_Q27H, H_S56H / L_Q27H, H_S53&56H / L_Q27H, parent antibody Lecanemab and anti-His antibody as a negative control to Aβ protofibrils under near-neutral or acidic pH conditions are shown;

[0027] Figure 4 The graph showing the binding force of exemplary mutant antibodies H_S53H / L_Q27H, H_S56H / L_Q27H, parent antibody Lecanemab and anti-His antibody as a negative control to Aβ protofibrils under heating or room temperature conditions is shown;

[0028] Figure 5Competitive binding of the parent antibody Lecanemab ( Figure 5 A) or exemplary mutant antibody H_S56H / L_Q27H ( Figure 5 B) ELISA test results;

[0029] Figure 6 The parent antibody Lecanemab ( Figure 6 A) and an exemplary mutant antibody of the present invention H_S56H / L_Q27H ( Figure 6 B) Sandwich ELISA results for Aβ protofibrils, Aβ(1-42) peptide, and Aβ(1-16) peptide monomers. DETAILED DESCRIPTION

[0030] definition

[0031] As used in the description of the present invention, the following words and phrases are generally deemed to have the meanings set forth below, unless otherwise indicated in the context in which the words or phrases are used.

[0032] As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. As used herein, the terms "comprising", "including", and "having" mean that compositions and methods include the recited components or steps, but do not exclude other components or steps.

[0033] As used herein, the term "about" refers to the common error range of the corresponding value that is readily known to those skilled in the art. For example, relative to the described value, ±10% or less, ±5% or less, ±2% or less, ±1% or less, or ±0.1% or less. The value or parameter described in this article in the manner of "about" includes the value or parameter itself.

[0034] The term "Aβ protofibril" as used herein is a large molecular weight oligomeric form of Aβ peptide aggregated from soluble Aβ peptide monomers, which is soluble in vitro, about 670 kDa. Aβ protofibrils further aggregate to form insoluble fibrils, which then merge into amyloid plaques.

[0035] The term "antibody" as used herein refers to an immunoglobulin molecule composed of four polypeptide chains, namely two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HCVR or V H ) and heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2 and CH3. Each light chain contains a light chain variable region (LCVR or V L ) and a light chain constant region. The light chain constant region comprises a domain (CL1).H and V L The V domains can be further divided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged from amino-terminal to carboxyl-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "complementarity determining region" or "CDR" refers to the region of the antibody variable region that is primarily responsible for binding to the epitope of an antigen. The positioning and alignment of the CDRs can be performed in the three-dimensional space formed by the antibody framework regions, and the amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, such as those described by Kabat et al. (see Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273, 927-948, 1997; “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8: el002388, 2012; and Kunik et al., Nucleic Acids Res 40 (Web Server No. 5001574, 2013). issue): W521-524, 2012; "Paratome CDRs") and ImMunoGeneTics (IMGT) databases (see Lefranc, Nucleic Acids Res 29:207-9, 2001; "IMGT" numbering scheme). Kabat, Paratome and IMGT databases are all public databases on the Internet. The numbering rules for amino acids in the present invention adopt the Kabat numbering rules.

[0036] The term "functional fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as human Aβ protofibrils). The functional fragment of an antibody can be realized by certain fragments of a full-length antibody. Examples of binding fragments covered by the term "functional fragment" of an antibody include (i) a Fab fragment, i.e., a fragment consisting of V L 、V H(ii) a Fab' fragment; (iii) a F(ab')2 fragment, which is a bivalent fragment consisting of two Fab' fragments linked by a disulfide bond in the hinge region; and (iv) a fragment consisting of V H and CH1 domains; (v) a single-arm V L and V H Fv fragment composed of V H dAb fragments composed of all or part of the domains (Ward et al. (1989) Nature 241: 544-546); and (vi) CDR. In addition, although the two domains V L and V H are encoded by different genes, but they can be linked together by a synthetic linker through recombinant methods to form a single connected chain, in which V L and V H The single-chain Fv (scFv) is a monovalent molecule formed by pairing the two domains of the antibody with each other (see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also included in the term "functional fragment" of an antibody. Other forms of single-chain antibodies, such as bispecific antibodies, are also included (see, for example, Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90: 6444-6448).

[0037] The term sequence "identity" refers to the percentage of amino acid (or nucleotide) residues of the target sequence that are identical to the amino acid (or nucleotide) residues of the reference sequence after alignment and introduction of gaps. When calculating identity, any conservative substitution is not considered as part of sequence identity. Specifically, sequence identity can be calculated by the following method: the two sequences are matched in a comparison window as an optimal alignment, the number of positions matched in the two sequences is divided by the total number of positions in the comparison window (i.e., the window size), and the result is then multiplied by 100. Alignment can be achieved by alignment methods known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR). Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence.

[0038] The term "expression vector" refers to a vector that can transform a host cell and contains a nucleic acid sequence that directs and / or controls (together with the host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of a coding region operably linked thereto.

[0039] The host cell can be any prokaryotic or eukaryotic cell containing an expression vector. The term also includes those prokaryotic or eukaryotic cells, their progeny, and transgenic plants or transgenic animals composed of or containing the cells that have been genetically engineered to contain cloned target genes in the chromosome or genome of the host cell, or in the cells of the host cell.

[0040] Herein, His is the abbreviation of histidine. Unless otherwise specified, histidine in the present invention relates to all its isomeric forms, including but not limited to L-histidine or D-histidine.

[0041] "Treatment" or "treatment" refers to clinical intervention in an attempt to alter the disease in the individual being treated. The desired effects of treatment include, but are not limited to, preventing recurrence of the disease, alleviating symptoms, alleviating / reducing any indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis.

[0042] As used herein, the term "pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0043] Herein, the term "effective amount" means an amount that can produce the desired effect on the subject. The exact dosage will depend on the purpose of treatment, the age and size of the subject, the route of administration, etc., and can be determined by professionals in the field using known techniques (e.g., see Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0044] Herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. In a preferred case, the subject is a human.

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Chain Reaction" (Mullis et al., 1994); and Current Protocols in Immunology (JE Coligan et al., 1991), each of which is expressly incorporated herein by reference. The following is a description of the preferred mode for implementing the present invention. It should be noted that the embodiments described below are examples of representative embodiments of the present invention, but the present invention is not limited to these embodiments.

[0047] The experimental equipment and reagents involved in the following examples are shown in Table 1 and Table 2.

[0048] Table 1 Experimental equipment

[0049]

[0050] Table 2 Reagents

[0051]

[0052] In the following examples, PBST is prepared by adding Tween 20 to 1×PBS (pH 7.4), wherein the amount of Tween 20 added is 0.05% of 1×PBS, such as adding 0.05 g of Tween 20 to 100 mL of 1×PBS and fully dissolving it.

[0053] PBST containing 1% BSA was prepared by adding 0.1 g of BSA to 10 mL of PBST and fully dissolving it before use.

[0054] The pH 7.4 diluent is PBST solution containing 1% BSA.

[0055] The pH 5.0 diluent is 0.15 M citrate phosphate solution containing 1% BSA and 0.05% Tween 20.

[0056] Amplex Red developing solution: H2O2 and phosphate buffer (30 mM, pH 7.2) were mixed at a ratio of 1:3000 to obtain a mixed solution, and then Amplex Red reagent was mixed with the above mixed solution at a ratio of 1 g:750 mL.

[0057] Aβ protofibril solution: 50 μg of freeze-dried Aβ(1-42) was fully dissolved in 110 μl of 2 mM NaOH solution, and then an equal volume (110 μl) of F12 medium was added, mixed gently and thoroughly, and incubated at 37°C for 72 hours. Then, centrifuged at 16000 g for 5 minutes to remove possible fiber precipitation, and the supernatant was aspirated as the Aβ protofibril solution.

[0058] Biotin-labeled antibody: Take 200-500 μL of purchased antibody solution in a manner containing 1 mg of antibody, and add PBS until the volume of the antibody solution becomes 500 μL. Calculate the amount of 10mM EZ-Link sulfo-NHS-LC-Biotin according to the following formula: (1mg / molecular weight of antibody)*20*100000=VBiotin, add VBiotin 10mM EZ-Link sulfo-NHS-LC-Biotin to the antibody solution, mix thoroughly, and let stand at room temperature for 30 minutes for biotin labeling. Afterwards, desalt with a desalting column according to the instructions, and remove the unlabeled antibody to obtain a purified biotin-labeled antibody.

[0059] Example 1

[0060] This example explores the changes in the binding ability of Lecanemab antibody mutants to Aβ protofibrils after the introduction of histidine mutations.

[0061] 1. Cloning and site-directed mutagenesis

[0062] Synbio was commissioned to synthesize the coding nucleotide sequences of the heavy chain and light chain of the parent antibody (SEQ ID NO: 36 and 37), and cloned into the SalI restriction enzyme site BglII of the pcDNA vector (Ab Studio) to obtain recombinant vectors pcDNA-Lecanemab-HC and pcDNA-Lecanemab-LC expressing the heavy chain and light chain of the parent antibody, respectively. Using the QuikChange II site-directed mutagenesis kit (purchased from Agilent), site-directed mutations were introduced at different sites of the coding sequence to obtain a series of recombinant vectors, which respectively encode His point mutations (hereinafter also referred to as V H Variant or V L Specifically, the obtained recombinant vector comprises the following coding sequence:

[0063] Table 3

[0064]

[0065]

[0066] 2. Transient transfection

[0067] The combinations of the seven antibody heavy chain recombinant vectors and light chain recombinant vectors shown in Table 3 were transiently transfected into 293 cells. After 72 hours, the supernatant was collected and the antibodies were purified using HiTrap Protein AHP (cytiva, #17040301) antibody purification chromatography column. The purified antibodies had the following CDR combinations.

[0068] Table 4

[0069]

[0070] 3. Determination of the binding affinity of the parental antibody and different mutants to Aβ protofibrils by ELISA

[0071] It has been reported that when Aβ(1-42) polypeptide is attached to an ELISA plate, its ability to capture Lecanemab antibody is similar to the ability of Aβ protofibrils in solution to capture Lecanemab antibody. Coat a high binding ELISA plate with 50 μL / well of PBS solution containing 1 μg / mL of human amyloid β fragment (1-42). Cover the ELISA plate and incubate overnight at 4°C. Discard the coating solution and wash 6 times with PBST. Then, add 100 μL of PBST containing 1% BSA to each well and incubate at room temperature for 1 hour. Discard the solution and wash 6 times with PBST. Then, dilute the antibody gradient to 500, 50, 5, 0.5 or 0.05 ng / mL with pH 7.4 diluent, add 50 μL of antibody diluent to each well, and incubate at room temperature for 1 hour. Discard the solution and wash 6 times with PBST. Dilute HRP-conjugated goat anti-human IgGFc secondary antibody 1:5000 in pH 7.4 diluent, add 50 μL to each well, and incubate at room temperature for 1 hour. Discard the solution and wash 6 times with PBST. Add 50 μL of Amplex Red developing solution to each well and immediately place it in a fluorescent microplate reader. Read the plate using an excitation spectrum (Ex) of 530 nm, an emission spectrum (Em) of 390 nm, and a cutoff setting of 570 nm. Detect the fluorescence value after 2 minutes of development.

[0072] The results obtained from the parent antibody and exemplary heavy chain mutant antibodies (H_T28E, H_S53H, H_S56H, H_Y50H, H_Y109H) are as follows: Figure 1 As shown. Figure 1 It can be seen that compared with the parent antibody, the H_Y50H mutant almost lost the ability to bind to Aβ protofibrils; the H_Y109H and H_T28E mutants were still able to bind to Aβ protofibrils, but the affinity was significantly weaker than that of the parent antibody; the H_S53H and H_S56H mutants showed Aβ protofibril binding affinity that was basically the same as that of the parent antibody, among which the H_S53H mutant was the best.

[0073] The results obtained from the parent antibody and exemplary light chain mutant antibodies (L_Q27H, L_S28H) are as follows: Figure 2 As shown. Figure 2 It can be seen that compared with the parent antibody, the binding affinity of the L_S28H mutant to Aβ protofibrils was significantly weakened, but the L_Q27H mutant showed a slightly stronger binding affinity than the parent antibody.

[0074] Example 2

[0075] Based on the binding of the histidine single mutant to the target antigen, double mutants and triple mutants having histidine mutations in both the heavy chain and the light chain were further prepared, as shown in Table 5 below.

[0076] Table 5

[0077]

[0078] The parent antibody (Lecanemab), mutant antibodies (including double mutants and triple mutants) and negative control antibody (anti-His antibody) were diluted to the following concentrations with pH 7.4 diluent and pH 5.0 diluent, respectively: 10000, 1000, 100, 10, 1, 0.1, 0.01 ng / mL. The binding affinity curves of the parent antibody and each mutant antibody to Aβ protofibrils under pH 7.4 and pH 5.0 were determined by the same ELISA method as in Example 1. The results are shown in Figure 3 and Table 6.

[0079] Table 6

[0080]

[0081] It can be seen that under a physiological pH close to neutral (pH 7.4), each mutant of the present invention has almost the same binding affinity as the parent antibody, which means that it can bind tightly to the target antigen in vivo; and under acidic pH conditions simulating the inside of lysosomes (pH 5.0), the mutant antibodies of the present invention all have EC50 values ​​significantly higher than those of the parent antibody, indicating that after entering the lysosome as an antigen-antibody complex, the mutant antibodies of the present invention will be able to dissociate from the antigen more easily than the parent antibody, and be recycled back to the cell through the antibody and used again for the degradation of the target antigen, thereby achieving more favorable pharmacokinetics (such as extended half-life, reduced in vivo clearance, etc.).

[0082] Example 3

[0083] This example demonstrates that the mutants of the invention are thermostable in binding to Aβ protofibrils.

[0084] The binding affinity between the antibody and the antigen was determined by an ELISA method substantially the same as in Example 1, except that after blocking with PBST containing 1% BSA at room temperature for 60 minutes, the cells were washed 4 times with PBST, and the parent antibody (Lecanemab), mutant antibodies (including H_S53H / L_Q27H double mutants, H_S56H / L_Q27H double mutants) and negative control antibodies (anti-His antibodies) were diluted with pH 7.4 diluent to the following concentrations: 10000, 1000, 100, 10, 1, 0.1 or 0.01 ng / mL, and then each sample was heated at 70°C for 5 minutes and at room temperature for 5 minutes without heating, and then 50 μL of the treated antibody sample dilution was added to different ELISA plate wells, respectively. The cells were incubated on a plate shaker at room temperature for 60 minutes. The cells were washed four times with PBST, and HRP-conjugated goat anti-human IgG Fc secondary antibody was added at a dilution ratio of 1:5000 in a standard pH 7.4 diluent, and the readings were measured under the same conditions as in Example 1. The results are shown in Figure 4 shown.

[0085] from Figure 4 It can be seen that no reduction in binding was observed for all combined mutations under heating or non-heating conditions, and the binding force was consistent with that of the parent.

[0086] Example 4

[0087] In this embodiment, the binding ability of the His double mutant of the present invention to Aβ protofibrils or Aβ monomers was measured by competitive ELISA.

[0088] Coat the high binding ELISA plate with 0.5 μM Aβ protofibril solution at 50 μL / well. Cover the ELISA plate and incubate overnight at 4°C. Discard the coating solution and wash 6 times with PBST. Then, add 1% BSA in PBST at 100 μL / well and incubate at room temperature for 1 hour for blocking. Discard the blocking solution and wash 6 times with PBST for later use.

[0089] The Aβ protofibril solution was diluted 3-fold with pH 7.4 diluent to the following concentrations: 1000.000, 333.333, 111.111, 37.037, 12.346, 4.115, 1.372, 0.457, 0.152, 0.051, 0.017 or 0.006 nM; the Aβ1-16 monomer solution was diluted 3-fold with pH 7.4 diluent to the following concentrations: 25000.000, 8333.333, 2777.778, 925.926, 308.642, 102.881, 34.294, 11.431, 3.810, 1.270, 0.423 or 0.141 nM. Add 30 μL of the above gradient diluted Aβ protofibril solution or Aβ(1-16) monomer solution to each well of a low adsorption 96-well plate, and at the same time, add 30 μL of the parent antibody (Lecanemab) or H_S56H / L_Q27H double mutant at a concentration of 0.1 μg / mL to each well. Incubate at 900 rpm and room temperature for 45 minutes. Then, transfer the supernatant of each well to different wells of the ELISA plate coated with Aβ protofibrils and blocked, and incubate at room temperature for 25 minutes. Discard the solution and wash 6 times with PBST. Add 50 μL of HRP-coupled goat anti-human IgG Fc secondary antibody diluted 1:10000 in pH 7.4 diluent in advance to each well and incubate at room temperature for 1 hour. Discard the solution and wash 6 times with PBST. Finally, 50 μL of TMB colorimetric solution was added to each well. After 2 min of development, the reaction was terminated with TMB colorimetric stop solution. The absorbance of each well solution was measured at a wavelength of 450 nm using an ELISA reader. The results were displayed on Figure 5 .

[0090] from Figure 5 It can be seen that when co-incubated with Aβ protofibrils, as the concentration of co-incubated Aβ protofibrils increases, the amount of the parent antibody (Lecanemab) and H_S56H / L_Q27H double mutant bound to the Aβ protofibrils coated on the ELISA plate is significantly reduced; in contrast, when co-incubated with Aβ1-16 monomers, the amount of Lecanemab and H_S56H / L_Q27H double mutant bound to the Aβ protofibrils coated on the ELISA plate is almost unchanged. This indicates that the H_S56H / L_Q27H double mutant has recognition specificity and strong affinity for Aβ protofibrils, and its binding affinity for Aβ protofibrils is significantly higher than that for Aβ(1-16) monomers, and the degree of difference is consistent with that of the parent antibody.

[0091] Example 5

[0092] In this example, the ability of the His double mutant of the present invention to bind to Aβ protofibrils, Aβ polypeptides, and Aβ monomers was evaluated by sandwich ELISA.

[0093] Coat the high binding ELISA plate with 0.125 μg / mL of parent antibody (Lecanemab) in PBS or H_S56H / L_Q27H double mutant in PBS at 50 μL / well. Cover the ELISA plate and incubate overnight at 4°C. Discard the coating solution and wash 6 times with PBST. Then, add PBST containing 2% BSA at 100 μL / well and incubate at room temperature for 1 hour. Discard the solution and wash 6 times with PBST for later use.

[0094] Using PBST containing 0.1% BSA as a diluent, prepare Aβ protofibril solutions, Aβ(1-42) polypeptide solutions, and Aβ(1-16) monomer solutions at concentrations of 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or 0.000001 μM, respectively. Add the diluted sample solutions to the ELISA plates coated with the parent antibody or double mutant antibody at 100 μL per well, and incubate at room temperature for 2 hours. Discard the solution and wash with PBST 6 times. Then, add the biotin-labeled parent antibody (Lecanemab) or the biotin-labeled H_S56H / L_Q27H double mutant diluted in advance with PBST containing 0.5% BSA (1:10000) and incubate at room temperature for 1 hour. Discard the solution and wash with PBST 6 times. Finally, 50 μL of TMB colorimetric solution was added to each well. After 2 min of development, the reaction was terminated with TMB colorimetric stop solution. The absorbance of each well solution was measured at a wavelength of 450 nm using an ELISA reader. The results were displayed on Figure 6 .

[0095] Figure 6 The sandwich ELISA results showed that the H_S56H / L_Q27H double mutant selectively binds to Aβ polypeptides in different aggregation states, that is, it has the strongest affinity for Aβ protofibrils, the second strongest affinity for Aβ(1-42), and almost no affinity for Aβ(1-16) monomers, which is consistent with the parent antibody.

[0096] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further modifications may be made to the present invention. In short, the present invention is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of the present invention, in accordance with the principles of the present invention. Applications of some of the basic features may be made within the scope of the claims appended hereto.

Claims

1. An Aβ protofibril antibody or a functional fragment thereof, characterized in that: It has at least one histidine mutation relative to the amino acid sequence of lencanezumab and comprises: (a) an antibody heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 2, 3, 4, 6 or 7, or a combination of VH-CDR1, VH-CDR2 and VH-CDR3 in an antibody heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 19, 20, 22, 26 or 28; and / or (b) an antibody light chain having the amino acid sequence shown in SEQ ID NO: 9 or 10, or a combination of VL-CDR1, VL-CDR2 and VL-CDR3 in the variable region of an antibody light chain having the amino acid sequence shown in SEQ ID NO: 30 or 32.

2. The Aβ protofibril antibody or a functional fragment thereof according to claim 1, characterized in that: It comprises a CDR combination selected from any one of (a) to (q): (a) VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, A VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 13, A VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 14, A VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 16, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (b) VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, A VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 21, A VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 14, A VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 16, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (c) a VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, A VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 23, A VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 14, A VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 16, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (d) a VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 13, a VH-CDR3 having the amino acid sequence of SEQ ID NO: 27, a VL-CDR1 having the amino acid sequence of SEQ ID NO: 16, a VL-CDR2 having the amino acid sequence of SEQ ID NO: 17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 18; (e) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 12, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 29, a VH-CDR3 having the amino acid sequence of SEQ ID NO: 14, a VL-CDR1 having the amino acid sequence of SEQ ID NO: 16, a VL-CDR2 having the amino acid sequence of SEQ ID NO: 17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 18; (f) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO:12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO:13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO:14, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO:31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO:17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO:18; (g) a VH-CDR1 having the amino acid sequence of SEQ ID NO:12, a VH-CDR2 having the amino acid sequence of SEQ ID NO:13, a VH-CDR3 having the amino acid sequence of SEQ ID NO:14, a VL-CDR1 having the amino acid sequence of SEQ ID NO:33, a VL-CDR2 having the amino acid sequence of SEQ ID NO:17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO:18; (h) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 12, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 21, a VH-CDR3 having the amino acid sequence of SEQ ID NO: 14, a VL-CDR1 having the amino acid sequence of SEQ ID NO: 31, a VL-CDR2 having the amino acid sequence of SEQ ID NO: 17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 18; (i) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 12, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 23, a VH-CDR3 having the amino acid sequence of SEQ ID NO: 14, a VL-CDR1 having the amino acid sequence of SEQ ID NO: 31, a VL-CDR2 having the amino acid sequence of SEQ ID NO: 17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 18; (j) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 12, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 29, a VH-CDR3 having the amino acid sequence of SEQ ID NO: 14, a VL-CDR1 having the amino acid sequence of SEQ ID NO: 31, a VL-CDR2 having the amino acid sequence of SEQ ID NO: 17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 18; (k) a VH-CDR1 having the amino acid sequence of SEQ ID NO:12, a VH-CDR2 having the amino acid sequence of SEQ ID NO:13, a VH-CDR3 having the amino acid sequence of SEQ ID NO:14, a VL-CDR1 having the amino acid sequence of SEQ ID NO:31, a VL-CDR2 having the amino acid sequence of SEQ ID NO:17, and a VL-CDR3 having the amino acid sequence of SEQ ID NO:18; (1) a VH-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a VH-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 27, a VL-CDR1 having an amino acid sequence as set forth in SEQ ID NO: 31, a VL-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, and a VL-CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18; (m) a VH-CDR1 having the amino acid sequence of SEQ ID NO: 12, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 13, a VH-CDR3 having the amino acid sequence of SEQ ID NO: 14, and a VL-CDR1 having the amino acid sequence of SEQ ID NO: 33, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (n) a VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, A VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 21, A VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 14, A VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 33, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (o) VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, A VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 23, A VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 14, A VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 33, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (p) VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, A VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 13, A VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 27, A VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 33, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO: 18; (q) VH-CDR1 having the amino acid sequence shown in SEQ ID NO: 12, A VH-CDR2 having the amino acid sequence shown in SEQ ID NO: 29, A VH-CDR3 having the amino acid sequence shown in SEQ ID NO: 14, A VL-CDR1 having the amino acid sequence shown in SEQ ID NO: 33, A VL-CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and A VL-CDR3 having the amino acid sequence shown in SEQ ID NO:

18.

3. The Aβ protofibril antibody or a functional fragment thereof according to claim 1 or 2, characterized in that: It comprises an antibody heavy chain having an amino acid sequence as shown in any one of SEQ ID NO: 2, 3, 4, 6 or 7, or an antibody heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 19, 20, 22, 26 or 28, and / or an antibody light chain having an amino acid sequence as shown in SEQ ID NO: 9 or 10, or an antibody light chain variable region having an amino acid sequence as shown in SEQ ID NO: 30 or 32.

4. The Aβ protofibril antibody or a functional fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody further comprises a constant region; optionally, the constant region is an IgG constant region, optionally, the constant region is selected from the constant region of IgG1 or IgG4; Optionally, the light chain constant region comprised by the antibody has an amino acid sequence as shown in SEQ ID NO: 35, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having one or more site substitutions, deletions or insertions compared to the amino acid sequence shown in SEQ ID NO: 35; and / or The heavy chain constant region contained in the antibody has an amino acid sequence as shown in SEQ ID NO:34, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having one or more site substitutions, deletions or insertions compared to the amino acid sequence shown in SEQ ID NO:

34.

5. The Aβ protofibril antibody or a functional fragment thereof according to any one of claims 1 to 4, characterized in that: The functional fragment is selected from any one of Fab, Fab', F(ab')2, Fd, Fv, scFv and dAb.

6. The Aβ protofibril antibody or a functional fragment thereof according to any one of claims 1 to 5, characterized in that: It also comprises an element or modification that increases protein stability, an element or modification that prolongs half-life, a separation or purification tag, an immune activation element and / or an in vivo delivery element; optionally, the in vivo delivery element is selected from a membrane-penetrating peptide, a nuclear localization signal, an organelle localization signal, an endoplasmic reticulum retention signal, a peroxisome targeting signal and / or a mitochondrial transit peptide. 7 . A nucleic acid molecule encoding the Aβ protofibril antibody or a functional fragment thereof according to any one of claims 1 to 6 .

8. An expression vector comprising the nucleic acid molecule according to claim 7, Optionally, the expression vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector.

9. A recombinant virus particle comprising the expression vector according to claim 8.

10. A host cell expressing the Aβ protofibril antibody or a functional fragment thereof according to any one of claims 1 to 6, or comprising the nucleic acid molecule according to claim 7 or the expression vector according to claim 8, or expressing the recombinant virus particle according to claim 9.

11. A composition comprising the Aβ protofibril antibody or a functional fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant virus particle according to claim 9 or the host cell according to claim 10; Optionally, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable excipient.

12. Use of the Aβ protofibril antibody or its functional fragment according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant virus particle according to claim 9, the host cell according to claim 10 or the composition according to claim 11 in the preparation of a drug for treating or preventing a disease caused by aggregation or deposition of β-amyloid protein; Optionally, the disease is a neurological disease, Optionally, the disease is selected from traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, Parkinson's disease dementia (PDD) or Alzheimer's disease (AD); Optionally, the disease is Alzheimer's disease; Optionally, the disease is mild cognitive impairment caused by Alzheimer's disease and mild dementia caused by Alzheimer's disease.

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