Development and use of therapeutic agent for alzheimer's disease

Abstract

Provided is an antibody that binds to β-amyloid (Aβ), or an antigen-binding fragment thereof. Further provided are a nucleic acid encoding the antibody or the antigen-binding fragment thereof, a cell comprising the antibody or the antigen-binding fragment or nucleic acid thereof, a pharmaceutical composition, a kit, and the use of the antibody or the antigen-binding fragment thereof in the preparation of a drug used for treating or preventing a disease caused by abnormal accumulation or deposition of Aβ in subjects.

Description

Development and application of therapeutic agents for Alzheimer's disease

[0001] This application claims priority to Chinese patent application No. 202311083911.0 filed on August 25, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0002] The present invention belongs to the field of biomedicine, and in particular relates to antibodies binding to beta-amyloid protein (beta-Amyloid, Aβ) and applications thereof. Background Art

[0003] Alzheimer's disease (AD) is a neurodegenerative disorder that occurs frequently in the elderly, accounting for 50% to 70% of dementia cases worldwide and the most common form of dementia. It is a physical brain disease characterized by impaired brain function, such as memory, language, thinking, and behavior. It is a progressive disease caused by the gradual degeneration of brain cells. Clinically, it is characterized by progressive memory and cognitive impairment, as well as behavioral abnormalities. Typical pathological manifestations include amyloid deposits, neurofibrillary tangles (NFTs), neuronal loss, axonal and synaptic abnormalities, and granulocytic vacuolar degeneration. With the increasing aging of the population, the incidence of AD is steadily increasing, and its medical care and care impose a heavy economic and psychological burden on society and families. Even after adjusting for other risk factors, the relative risk of death associated with AD remains higher than that of other types of dementia, particularly in people under 75 years of age.

[0004] The pathogenesis of Alzheimer's disease remains unclear, with the most prominent theories being the amyloid-β hypothesis and the Tau protein hypothesis. The amyloid cascade hypothesis has long been a leading explanation for the pathogenesis of Alzheimer's disease (Hardy JA et al., Science, (1992), 368:387-403). This hypothesis posits that abnormal metabolism of amyloid protein precursor (APP) in the brain increases Aβ production and decreases degradation, leading to massive Aβ accumulation. Excessive Aβ accumulation forms amyloid plaques, which produce neurotoxicity.

[0005] The primary cause of Aβ deposition in brain tissue is abnormal Aβ anabolism and decreased catabolism, leading to an imbalance in Aβ transport. Aβ anabolism primarily involves α-, β-, and γ-secretases. α-secretase inhibits Aβ production, while β- and γ-secretases promote Aβ anabolism. β-secretase, also known as amyloid precursor protein β-site cleaving enzyme-1 (BACE-1), is a critical, rate-limiting enzyme in the Aβ production process. Therefore, the BACE-1 gene is currently a hot topic in Alzheimer's disease research, but inhibitors currently developed for BACE-1 have all failed in clinical trials.

[0006] Excessive Aβ is currently considered a necessary, but not sufficient, trigger for Alzheimer's disease. Targeting Aβ as a therapeutic agent for AD has become a major focus of clinical research. Aβ deposition can contribute to the onset of Alzheimer's disease, and reducing Aβ accumulation in brain tissue can delay or alleviate Alzheimer's symptoms. Therefore, understanding how to eliminate Aβ and improve cognition is crucial.

[0007] Over the past three decades, the vast majority of anti-Aβ antibody drug development efforts by major pharmaceutical companies have failed. In August 2012, Pfizer and Johnson & Johnson announced the suspension of further research on the anti-Aβ monoclonal antibody bapineuzumab. In a previously conducted Phase III clinical trial, bapineuzumab failed to improve patients' memory loss and failed to reach the clinical endpoint, resulting in the clinical study being declared a failure. In December 2014, Roche's heavily invested Alzheimer's disease drug gantenerumab failed in Phase III clinical trials. On November 23, 2016, Eli Lilly and Company announced the results of the EXPEDITION 3 trial: solanezumab failed to achieve the expected efficacy, thus declaring the $3 billion Alzheimer's disease candidate a failure. On January 30, 2019, Roche announced the termination of its two Phase III CREAD I and CREAD 2 clinical studies of crenezumab in patients with early Alzheimer's disease (prodromal or mild AD). On November 6, 2020, the FDA expert committee voted on the clinical research evidence of Biogen's Aducanumab, and believed that the EMERGE trial could not provide evidence of effectiveness, and also believed that the PRIME trial could not provide evidence of effectiveness. However, on June 7, 2021, the FDA officially approved Biogen's Aducanumab for the treatment of Alzheimer's disease, and its trade name is Aduhelm. However, after it was launched on the market, patients did not benefit much and there was a serious risk of cerebral edema. On January 6, 2023, the FDA approved the new Alzheimer's disease drug lecanemab-irmb jointly developed by Eisai and Biogen through an accelerated approval pathway. Lecanemab can alleviate cognitive decline to a certain extent, but it is also accompanied by the risk of cerebral edema. Donanemab developed by Eli Lilly has shown good Aβ plaque clearance effects in Phase II and early Phase III clinical trials and is expected to be launched in the future.

[0008] Years of development experience, lessons learned, and achievements have demonstrated that antibody drugs targeting Aβ can clear Aβ from the brain, alleviate cognitive decline, and improve brain pathological symptoms. However, while the Aβ antibody drugs developed by Eisai and Eli Lilly can alleviate cognitive decline to a certain extent, they still face issues with their half-life and safety. For example, in the Phase I clinical trial of Lecanemab, the half-life of the 10mg / kg group was 6.9 days. In the Phase I / II clinical trial of Donanemab, the half-life of the intravenously administered 1mg / kg group was 3.19 days, and the half-life of the 10mg / kg group was 10.5 days. Studies have shown that Alzheimer's patients require long-term medication. To enhance the convenience of antibodies and reduce antibody dosage, the development of long-acting anti-Aβ antibodies holds great promise.

[0009] Therefore, since monoclonal antibody drugs with longer half-lives may achieve better efficacy, improvements in Aβ antibodies are still necessary. There is an urgent need to develop a new generation of antibody drugs for Alzheimer's disease or other diseases caused by Aβ deposition.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention immunized mice with amyloid protofibrils or pyroglutamyl-modified β-amyloid protein, generating multiple high-affinity antibodies that recognize amyloid. Activity evaluations have shown that these antibodies can effectively clear amyloid, exhibit favorable PK parameters, low immunogenicity, and possess a long half-life, making them promising for the treatment of Alzheimer's disease and other diseases characterized by abnormal amyloid accumulation or deposition.

[0012] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof that binds to β-amyloid (Aβ) is provided, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0013] (1) the VH comprises a heavy chain complementarity determining region (HCDR) 1 as set forth in SEQ ID NO: 23, a HCDR2 as set forth in SEQ ID NO: 24, and a HCDR3 as set forth in SEQ ID NO: 25, and the VL comprises a light chain complementarity determining region (LCDR) 1 as set forth in SEQ ID NO: 28, a LCDR2 as set forth in SEQ ID NO: 29, and a LCDR3 as set forth in SEQ ID NO: 30;

[0014] (2) the VH comprises HCDR1 as set forth in SEQ ID NO: 69, HCDR2 as set forth in SEQ ID NO: 70, and HCDR3 as set forth in SEQ ID NO: 71, and the VL comprises LCDR1 as set forth in SEQ ID NO: 74, LCDR2 as set forth in SEQ ID NO: 248, and LCDR3 as set forth in SEQ ID NO: 76;

[0015] (3) the VH comprises HCDR1 as shown in SEQ ID NO: 69, HCDR2 as shown in SEQ ID NO: 70, and HCDR3 as shown in SEQ ID NO: 71, and the VL comprises LCDR1 as shown in SEQ ID NO: 74, LCDR2 as shown in SEQ ID NO: 75, and LCDR3 as shown in SEQ ID NO: 76;

[0016] (4) the VH comprises HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 5, and the VL comprises LCDR1 as shown in SEQ ID NO: 8, LCDR2 as shown in SEQ ID NO: 9, and LCDR3 as shown in SEQ ID NO: 10;

[0017] (5) the VH comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15, and the VL comprises LCDR1 as shown in SEQ ID NO: 18, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20;

[0018] (6) the VH comprises HCDR1 as shown in SEQ ID NO: 33, HCDR2 as shown in SEQ ID NO: 34, and HCDR3 as shown in SEQ ID NO: 35, and the VL comprises LCDR1 as shown in SEQ ID NO: 38, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20;

[0019] (7) the VH comprises HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43, and the VL comprises LCDR1 as shown in SEQ ID NO:46, LCDR2 as shown in SEQ ID NO:47, and LCDR3 as shown in SEQ ID NO:48;

[0020] (8) the VH comprises HCDR1 as shown in SEQ ID NO: 51, HCDR2 as shown in SEQ ID NO: 52, and HCDR3 as shown in SEQ ID NO: 53, and the VL comprises LCDR1 as shown in SEQ ID NO: 56, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20;

[0021] (9) the VH comprises HCDR1 as shown in SEQ ID NO: 59, HCDR2 as shown in SEQ ID NO: 60, and HCDR3 as shown in SEQ ID NO: 61, and the VL comprises LCDR1 as shown in SEQ ID NO: 64, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 66;

[0022] (10) the VH comprises HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 79, and the VL comprises LCDR1 as shown in SEQ ID NO: 82, LCDR2 as shown in SEQ ID NO: 83, and LCDR3 as shown in SEQ ID NO: 84;

[0023] (11) the VH comprises HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 88, and HCDR3 as shown in SEQ ID NO: 89, and the VL comprises LCDR1 as shown in SEQ ID NO: 92, LCDR2 as shown in SEQ ID NO: 93, and LCDR3 as shown in SEQ ID NO: 94;

[0024] (12) the VH comprises HCDR1 as shown in SEQ ID NO: 130, HCDR2 as shown in SEQ ID NO: 131, and HCDR3 as shown in SEQ ID NO: 279, and the VL comprises LCDR1 as shown in SEQ ID NO: 74, LCDR2 as shown in SEQ ID NO: 134, and LCDR3 as shown in SEQ ID NO: 76;

[0025] (13) the VH comprises HCDR1 as shown in SEQ ID NO: 59, HCDR2 as shown in SEQ ID NO: 137, and HCDR3 as shown in SEQ ID NO: 138, and the VL comprises LCDR1 as shown in SEQ ID NO: 141, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 66;

[0026] (14) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 144, and the VL comprises LCDR1 as shown in SEQ ID NO: 147, LCDR2 as shown in SEQ ID NO: 148, and LCDR3 as shown in SEQ ID NO: 149;

[0027] (15) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 123, and the VL comprises LCDR1 as shown in SEQ ID NO: 218, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 127;

[0028] (16) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 123, and the VL comprises LCDR1 as shown in SEQ ID NO: 126, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 127;

[0029] (17) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 103, and LCDR3 as shown in SEQ ID NO: 104;

[0030] (18) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 235, and LCDR3 as shown in SEQ ID NO: 104;

[0031] (19) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 238, and LCDR3 as shown in SEQ ID NO: 104;

[0032] (20) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 245, and LCDR3 as shown in SEQ ID NO: 104;

[0033] (21) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 102, LCDR2 as shown in SEQ ID NO: 103, and LCDR3 as shown in SEQ ID NO: 104;

[0034] (22) the VH comprises HCDR1 as shown in SEQ ID NO: 107, HCDR2 as shown in SEQ ID NO: 108, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 102, LCDR2 as shown in SEQ ID NO: 103, and LCDR3 as shown in SEQ ID NO: 111; or

[0035] (23) The VH comprises HCDR1 as shown in SEQ ID NO: 114, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 115, and the VL comprises LCDR1 as shown in SEQ ID NO: 102, LCDR2 as shown in SEQ ID NO: 118, and LCDR3 as shown in SEQ ID NO: 104.

[0036] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention bind to pyroE Aβ3-42 peptide. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention bind to Aβ1-42 protofibril filaments.

[0037] In some embodiments, the VH and VL of the antibody each comprise the following amino acid sequence:

[0038] (1) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 164 and SEQ ID NO: 219, respectively;

[0039] (2) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 166 and SEQ ID NO: 219, respectively;

[0040] (3) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 174 and SEQ ID NO: 219, respectively;

[0041] (4) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 176 and SEQ ID NO: 219, respectively;

[0042] (5) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 21 and SEQ ID NO: 26, respectively;

[0043] (6) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 197 and SEQ ID NO: 246, respectively;

[0044] (7) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 197 and SEQ ID NO: 249, respectively;

[0045] (8) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 67 and SEQ ID NO: 72, respectively;

[0046] (9) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 1 and SEQ ID NO: 6, respectively;

[0047] (10) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 11 and SEQ ID NO: 16, respectively;

[0048] (11) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 31 and SEQ ID NO: 36, respectively;

[0049] (12) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 39 and SEQ ID NO: 44, respectively;

[0050] (13) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 49 and SEQ ID NO: 54, respectively;

[0051] (14) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 57 and SEQ ID NO: 62, respectively;

[0052] (15) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 77 and SEQ ID NO: 80, respectively;

[0053] (16) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 85 and SEQ ID NO: 90, respectively;

[0054] (17) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 128 and SEQ ID NO: 132, respectively;

[0055] (18) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 135 and SEQ ID NO: 139, respectively;

[0056] (19) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 142 and SEQ ID NO: 145, respectively;

[0057] (20) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 160 and SEQ ID NO: 216, respectively;

[0058] (21) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 152 and SEQ ID NO: 216, respectively;

[0059] (22) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 156 and SEQ ID NO: 216, respectively;

[0060] (23) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 158 and SEQ ID NO: 216, respectively;

[0061] (24) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 162 and SEQ ID NO: 216, respectively;

[0062] (25) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 160 and SEQ ID NO: 214, respectively;

[0063] (26) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 152 and SEQ ID NO: 214, respectively;

[0064] (27) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 162 and SEQ ID NO: 214, respectively;

[0065] (28) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 119 and SEQ ID NO: 124, respectively;

[0066] (29) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 230, respectively;

[0067] (30) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 233, respectively;

[0068] (31) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 236, respectively;

[0069] (32) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 185 and SEQ ID NO: 239, respectively;

[0070] (33) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 241, respectively;

[0071] (34) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 243, respectively;

[0072] (35) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 95 and SEQ ID NO: 100, respectively;

[0073] (36) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 105 and SEQ ID NO: 109, respectively; or

[0074] (37) The VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 112 and SEQ ID NO: 116, respectively.

[0075] In some embodiments, the antibodies of the present invention are selected from murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. In some embodiments, the antibodies are selected from the following antibody isotypes: IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibodies are selected from the following antibody subtypes: IgG1, IgG2, IgG3, and IgG4.

[0076] In some embodiments, the antibodies of the present invention comprise an IgG1 constant region (Fc region). In some embodiments, the Fc region comprises amino acid substitutions that extend the half-life of the antibody. In some embodiments, the amino acid substitutions comprise L234A and L235A substitutions.

[0077] In some embodiments, an antibody of the invention comprises a heavy chain (HC) and a light chain (LC), wherein:

[0078] (1) the HC and LC comprise amino acid sequences that have 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 to SEQ ID NO: 275 and SEQ ID NO: 277, respectively; or

[0079] (2) the HC and LC comprise amino acid sequences 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 to SEQ ID NO: 271 and SEQ ID NO: 273, respectively.

[0080] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv) and ds-scFv.

[0081] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention, the antibody is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

[0082] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0083] In a second aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0084] In a third aspect of the present invention, a vector is provided, which comprises the nucleic acid molecule of the second aspect of the present invention.

[0085] In a fourth aspect of the present invention, a cell is provided, the cell comprising the nucleic acid molecule of the second aspect of the present invention or the vector of the third aspect of the present invention.

[0086] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising:

[0087] a) culturing the cell of the fourth aspect of the invention under conditions suitable for expression of the antibody or antigen-binding fragment thereof; and

[0088] b) isolating the antibody or antigen-binding fragment thereof from the culture medium and / or culture supernatant of the cell.

[0089] In the sixth aspect of the present invention, a pharmaceutical composition is provided, which comprises the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention or the cell of the fourth aspect of the present invention, and optionally, a pharmaceutically acceptable carrier or excipient.

[0090] In the seventh aspect of the present invention, a kit is provided, which comprises the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, the cell of the fourth aspect of the present invention or the pharmaceutical composition of the sixth aspect of the present invention, and instructions for use; optionally, the kit further comprises an administration device.

[0091] In the eighth aspect of the present invention, provided is the use of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, the cell of the fourth aspect of the present invention or the pharmaceutical composition of the sixth aspect of the present invention in the preparation of a medicament for treating or preventing a disease caused by abnormal accumulation or deposition of Aβ in a subject.

[0092] In the ninth aspect of the present invention, provided is the use of the antibody or antigen-binding fragment thereof of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, the cell of the fourth aspect of the present invention or the pharmaceutical composition of the sixth aspect of the present invention in the preparation of a drug for clearing or alleviating abnormal accumulation or deposition of Aβ in a subject.

[0093] In some embodiments of the eighth and ninth aspects of the present invention, the subject is suffering from a degenerative disease of the central nervous system. In some embodiments, the subject is suffering from Alzheimer's disease (AD), Parkinson's disease (PD), Down syndrome or cerebral amyloid angiopathy, or is at risk of suffering from AD, PD, Down syndrome or cerebral amyloid angiopathy. In some embodiments, the subject is suffering from preclinical AD or clinical AD, for example, prodromal AD, mild AD, moderate AD or severe AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] FIG1 shows the activity of anti-Aβ chimeric antibodies in promoting the phagocytosis of Aβ by microglia.

[0095] Figure 2 shows the binding activity of anti-Aβ chimeric antibodies to pyroE Aβ3-16 and Aβ1-42 protofibrils. 151N5 and 200O1 bind strongly to pyroE Aβ3-16 and weakly to Aβ3-16; 6B5 and 7C8 bind strongly to protofibrils.

[0096] Figure 3 shows the affinity test results of humanized antibodies 6B5 and 7C8. The affinities of multiple humanized antibodies of 6B5 and 7C8 did not change significantly compared with the parent antibodies.

[0097] Figure 4 shows the affinity test results of the humanized antibodies 200O1 and 151N5. The affinities of the humanized antibodies of 200O1 / 151N5 did not change significantly compared with the parental antibodies.

[0098] Figure 5 shows the phagocytic activity assay results of the humanized antibodies 200O1 and 7C8. Similar to their parental antibodies, 200O1 H1K1 and 7C8 H6K2 can effectively promote the clearance of amyloid protein in brain slices by microglia.

[0099] Figure 6 shows the half-life of 200O1, 7C8 humanized antibodies, and their variants in mice. The half-life of 200O1 H1K1 is comparable to that of donanemab, and the half-life of 200O1 H1K1 LA is 2.45 times that of donanemab. The half-life of 7C8 H6K2 is comparable to that of lecanemab, and the half-life of 7C8 H6K2 LA is 2.3 times that of lecanemab.

[0100] Figure 7 shows the phagocytic activity assay results of the 200O1 and 7C8 humanized antibody variants. Both 200O1 H1K1 LA and 7C8 H6K2 LA can effectively promote the phagocytosis of Aβ by microglia in brain slices.

[0101] Figure 8 shows the immunogenicity test results of 200O1 and 7C8 humanized antibody variants. The immunogenicity of 200O1 H1K1 LA and 7C8 H6K2 LA were both superior to Donanemab and Lecanemab.

[0102] Figure 9 shows the binding activity of 200O1 and 7C8 humanized antibody variants to negative screening cells. 200O1 H1K1 LA negative screening did not bind to cells overexpressing amyloid precursor protein APP, and 7C8 H6K2 LA bound to negative screening cells weaker than Lecanemab.

[0103] FIG10 shows that seven days after administration, the brain concentration of the 7C8 humanized antibody variant tends to be higher than that of Lecanemab, and the brain concentration of the 20001 humanized antibody variant is significantly higher than that of Donanemab.

[0104] FIG11 shows that after three months of administration to 5X FAD transgenic mice, 7C8 and 20001 humanized antibody variants were more effective than Lecanemab and Donanemab in reducing brain Aβ levels.

[0105] FIG12 shows that after 13 weeks of administration to 5X FAD transgenic mice, 7C8 humanized antibody significantly reduced the levels of soluble and insoluble Aβ in the brain.

[0106] FIG13 shows that after 13 weeks of administration to 6-week-old FAD 4T transgenic mice, 7C8 humanized antibody significantly reduced the levels of soluble and insoluble Aβ in the brain.

[0107] FIG14 shows that after 13 weeks of administration to 15-week-old FAD 4T transgenic mice, 7C8 humanized antibody significantly reduced the levels of soluble and insoluble Aβ in the brain.

[0108] Detailed Description of the Invention

[0109] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0110] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies and in some embodiments, reference to "an antibody" includes a plurality of antibodies, and so forth.

[0111] Unless otherwise stated or defined, the term “comprise” and variations such as “includes”, “comprising” and “having” will be understood to imply the inclusion of stated elements or steps or groups of elements or steps but not the exclusion of any other elements or steps or groups of elements or steps.

[0112] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items. For example, a composition comprising A and / or B can be interpreted as a composition comprising A, a composition comprising B, or a composition comprising A and B.

[0113] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and are optionally varied (+) or (-) in increments of 1.0 or 0.1, or by variations of + / - 15%, 10%, 5%, or 2%, as appropriate. It should be understood that all numerical designations are preceded by the term "about." It should also be understood that the agents described herein are exemplary only and that equivalents thereof are known in the art. As used herein, the term "about" when referring to a measurable value such as an amount or concentration, etc., is intended to include variations within 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the specified amount.

[0114] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically comprise a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (such as effector cells) and components of the complement system such as C1q (the first component in the classical pathway of complement activation). Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the antibody portion that binds to the antigen.

[0115] A "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that specifically bind to an antigenic epitope. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework (FR) and CDR regions from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0116] The amino acid arrangement of each VL and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of the Chothia Kabat CDRs, in which CDR-H1 is a composite of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition by Martin et al. (world wide web bioinfo.org.uk / abs).

[0117] In the present application, the amino acid sequences of the CDRs are all shown in accordance with the Kabat definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in this area by a variety of methods. In the technical solution of the present invention, the Combined definition rule comprising the Kabat definition and the Chothia definition can also be used to determine the amino acid residues in the variable domain sequence. The Combined definition rule combines the scope of the Kabat definition and the Chothia definition, and takes a larger scope based on this. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or its region (such as a variable region) should be understood to encompass the complementary determining region defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in the present invention is based on the sequence shown in the Kabat definition rule, the amino acid sequence corresponding to the definition rules of other CDRs should also be within the scope of protection of the present invention.

[0118] The term "antibody" as used herein should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit the desired biological activity.

[0119] As used herein, the term "monoclonal antibody" generally refers to the antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies in the cluster are identical, except for a small amount of natural mutations that may exist. Monoclonal antibodies are generally highly specific for a single antigenic site. And, different from conventional polyclonal antibody preparations (generally having different antibodies for different determinants), each monoclonal antibody is for a single determinant on the antigen. Except for their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture, and are not polluted by other immunoglobulins. The modifier "monoclonal" represents the feature of the antibody obtained from a substantially homogeneous antibody population, and is not interpreted as needing to produce antibodies by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared in hybridoma cell, or can be prepared by recombinant DNA method.

[0120] The term "bispecific antibody" should be understood in the context of the present invention as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a target. The term "bispecific antibody" as used herein should be understood in its broadest sense, including full-length bispecific antibodies and antigen-binding fragments thereof. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0121] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to induce an adverse immune response in a human individual than the parent (e.g., mouse-derived) antibody.

[0122] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulin. In the CDR region, small additions, deletions, insertions, replacements, or modifications of amino acids may also be permitted, as long as they still retain the ability of the antibody to bind to a specific antigen. Humanized antibodies may optionally comprise at least a portion of a human immunoglobulin constant region. "Humanized antibodies" retain antigenic specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally comprise a chimeric antibody derived from a sequence of a non-human immunoglobulin. In some cases, the CDR region residues in the human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) (such as mouse, rat, rabbit, or non-human primate) with desired properties, affinity, and / or ability. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, such as binding affinity.

[0123] As used herein, the term "fully human antibody" generally refers to antibodies expressed by animals by transferring human antibody-encoding genes into genetically engineered antibody gene-deficient animals. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in this area include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0124] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., Aβ of the present invention). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0125] Examples of the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a partial hinge region; (iv) an Fd fragment, which consists of the VH and CH1 domains; (v) an Fd' fragment, which has the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of the VH domain; (viii) a single complementarity determining region (CDR); and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by synthetic linkers using recombinant methods so that they can form a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes "straight-chain antibodies" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptides and any modified forms of the aforementioned fragments that retain antigen-binding activity form an antigen-binding region.

[0126] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0127] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Avidity is represented by the equilibrium constant (KD) for the dissociation of antigen and antibody, and is a measure of the binding strength between an antigenic determinant and the antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD. Avidity is a measure of the binding strength between an antibody and the associated antigen. Avidity relates to the affinity between an antigenic determinant and the antigen binding site of an antibody and the number of associated binding sites present on the antibody.

[0128] Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and various variations thereof known in the art.

[0129] In the present invention, the term "KD" generally refers to the equilibrium dissociation constant, which is the ratio of the dissociation rate constant (kdis, also known as "off-rate (koff)" or "kd") to the association rate constant (kon, also known as "binding rate (kon)" or "ka"). The association rate constant (kon), dissociation rate constant (kdis) and equilibrium dissociation constant (KD) can be used to represent the binding affinity of an antigen-binding protein (e.g., an antibody) to an antigen. Methods for determining association and dissociation rate constants are well known in the art and include, but are not limited to, biomembrane interferometry (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and protein chip technology. The affinity of a particular protein-protein interaction measured may be different if measured under different conditions (e.g., salt concentration, pH).

[0130] The term "epitope" refers to the site on an antigen to which an antibody binds. An epitope can be formed by continuous amino acids or by the non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are typically retained in the exposure to denaturing solvents, while epitopes formed by the tertiary folding (also referred to as conformational epitopes) are typically lost in the treatment of denaturing solvents. An epitope typically includes at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. An epitope defines the minimum binding site of an antibody and is therefore the specific target of an antibody or its antigen-binding fragment.

[0131] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or double-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting of, or consisting essentially of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0132] The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably and in their broadest sense refer to a polymeric form of two or more amino acid subunits, amino acid analogs, or peptidomimetics. "Protein," "peptide," "polypeptide," and "amino acid sequence" contain at least two amino acids, and there is no limitation on the maximum number of amino acids. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including D and L optical isomers and amino acid analogs.

[0133] As used herein, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, amino acid sequence, or protein. If the nucleic acid sequence is from genomic DNA, expression may include splicing of mRNA in a eukaryotic cell.

[0134] The term "encoding" when applied to a nucleic acid sequence refers to a nucleic acid sequence that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA and / or translated to produce a polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0135] The term "sequence identity" refers to the sequence similarity between two polypeptides or between two nucleic acid sequences. The percent identity can be determined by comparing positions in each sequence that can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The degree of identity between sequences depends on the number of shared matching positions. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 25% identity with one of the sequences of the present invention. The alignment and percent sequence identity of the nucleic acid or amino acid sequences provided herein can be determined by importing the nucleic acid or amino acid sequence into ClustalW (available from https: / / genome.jp / tools-bin / clustalw / ) and using ClustalW.

[0136] When referring to specific molecules, biological materials or cellular substances, the terms "equivalent" or "functional variant" can be used interchangeably and refer to those with minimal homology while still retaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polypeptide (e.g., an antibody or antigen-binding fragment thereof described herein); or polypeptides encoded by polynucleotides having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference polynucleotide (e.g., a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein).

[0137] Compared to the antibodies or antigen-binding fragments thereof described in the present invention, equivalents with one or more amino acid modifications are also encompassed within the scope of the present invention, provided that the one or more amino acid modifications do not affect or substantially do not affect the ability of the antibody or antigen-binding fragment thereof to specifically bind to the antigen. As used herein, amino acid modifications can be amino acid substitutions, amino acid deletions, or amino acid insertions. Amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions. Conservative substitutions (also referred to as conservative mutations, conservative substitutions, or conservative variations) are amino acid substitutions in proteins that change a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, "conservative substitutions" refer to amino acid residues that are replaced by another biologically similar residue. Examples of conservative substitutions include one hydrophobic residue such as isoleucine, valine, leucine, or methionine replacing another; or one charged or polar residue replacing another, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, etc. Other illustrative examples of conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and the like.

[0138] As used herein, the term "subject" generally refers to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). The term "primate" generally refers to monkey and ape species, and includes monkey species, such as monkeys from the genus Macaca (such as, cynomolgus monkeys (Macaca fascicularis) and / or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), and marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), and ape species, such as chimpanzees (Pan troglodytes), and also includes Homo sapiens.

[0139] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression by transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0140] As used herein, the term "cell" generally refers to an individual cell, cell line, or cell culture that may or has contained a plasmid or vector comprising a nucleic acid molecule according to the present invention, or that is capable of expressing an antibody or antigen-binding fragment thereof according to the present invention. The cell may include progeny of a single host cell. Due to natural, accidental, or deliberate mutations, the progeny cells may not necessarily be identical in morphology or genome to the original parent cell, but may be capable of expressing the antibody or antigen-binding fragment thereof described herein. The cell may be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell, or a myeloma cell). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell. As used herein, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a specific cell, but also the offspring of these cells.

[0141] The term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and that contains no additional ingredients that are unacceptably toxic to the subject to which the composition is administered. The composition is sterile. A "sterile" composition is sterile, or free of all living microorganisms and their spores.

[0142] The term "kit" generally refers to a packaged product comprising components for administering an antibody or antigen-binding fragment thereof of the present invention to treat or prevent abnormal accumulation or deposition of amyloid protein to treat or prevent related diseases caused by the abnormal accumulation or deposition of amyloid protein. The components of the kit may be contained in separate vials (i.e., a kit with separate parts) or provided in a single vial. The kit may include reagents such as buffers, protein stabilizing agents, signal generating systems (e.g., fluorescent signal generating systems), antibodies, control proteins, and test containers. The kit may also include instructions for implementing the method. In some embodiments of the present invention, the kit may also include an administration device that can administer the active pharmaceutical ingredient (e.g., antibody or antigen-binding fragment thereof) in the kit to a subject in a suitable manner.

[0143] As used herein, the term "administering" is intended to mean delivering a substance to a subject, such as an animal or a human. Administration can be performed in a single dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with factors such as the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. In some embodiments, single or multiple administrations may be performed, with the dosage level and pattern being selected by a physician, or in the case of pets and other animals, by a veterinarian.

[0144] The term "disease caused by abnormal accumulation or deposition of Aβ" refers to a disease characterized pathologically by Aβ deposits in the brain or cerebrovascular system. This includes diseases such as Alzheimer's disease, Down syndrome and cerebral amyloid angiopathy. The clinical diagnosis, staging or progression of Alzheimer's disease can be easily determined by a primary diagnostician or health care professional as a person skilled in the art by using known techniques and by observing the results. This typically includes some forms of brain plaque imaging, mental or cognitive assessment (such as Clinical Dementia Rating-summary of boxes, CDR-SB, Mini-Mental State Exam (MMSE) or Alzheimer's Disease Assessment Scale-Cognitive (ADAS-Cog)) or functional assessment (such as Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL)). Cognitive and functional assessments can be used to identify changes in a patient's cognition (eg, cognitive decline) and function (eg, functional decline).

[0145] As used herein, "clinical Alzheimer's disease" or "clinical AD" is the diagnostic stage of Alzheimer's disease. It includes conditions diagnosed as prodromal Alzheimer's disease, mild Alzheimer's disease, moderate Alzheimer's disease, and severe Alzheimer's disease. The term "preclinical Alzheimer's disease" or "preclinical AD" is a stage before clinical Alzheimer's disease, in which measurable changes in biomarkers (such as CSF Aβ42 levels or deposited brain plaques by amyloid PET) indicate that patients with Alzheimer's pathology progress to the earliest signs of clinical Alzheimer's disease. This is usually before symptoms such as memory loss and confusion become significant. Preclinical Alzheimer's disease also includes presymptomatic autosomal dominant carriers and patients at higher risk of developing AD by virtue of carrying one or two APOE e4 alleles.

[0146] As used herein, "treating" includes limiting, slowing down or stopping the progression or severity of a patient's existing symptoms, condition, disease or illness. As understood in the art, "treatment" is a method for obtaining a beneficial or desired result, including a clinical result. For purposes of the present invention, a beneficial or desired result may include alleviating the extent of one or more diseases or conditions, delaying or slowing the progression of a disease or condition, improving or alleviating the state of a disease or condition (whether partially or completely). The term "preventing" means prophylactically administering an antibody or antigen-binding fragment thereof of the present invention to an asymptomatic patient or a patient with preclinical Alzheimer's disease to prevent the onset or progression of the disease.

[0147] Anti-Aβ antibodies

[0148] The amyloid hypothesis is currently recognized as the primary cause of AD. This theory posits that Aβ deposited around neurons causes neuronal loss by inducing apoptosis. The β-sheet structure of Aβ molecules promotes its aggregation into insoluble fibrils, which are resistant to protease degradation and further form insoluble precipitates, resulting in neurotoxicity. Current preventive measures against Aβ neurotoxicity primarily focus on reducing Aβ production, promoting its degradation and clearance, and preventing its aggregation and deposition.

[0149] Aβ is formed by the cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase. There are many subtypes of Aβ, for example, Aβ1-42, Aβ1-40, pyroE Aβ3-42, etc. PyroE Aβ3-42 is a truncated form of Aβ that lacks the first two amino acid residues at the N-terminus of Aβ and has a pyroglutamate modification derived from glutamic acid at the third amino acid position. Studies have found that deposits in plaques of human patients are composed of a heterogeneous mixture of Aβ peptides, and pyroE Aβ3-42 has been shown to have aggressive aggregation properties and accumulates early in the deposition cascade.

[0150] The amino acid sequence of human Aβ1-42 is: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 280).

[0151] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to β-amyloid (Aβ), the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0152] (1) the VH comprises a heavy chain complementarity determining region (HCDR) 1 as set forth in SEQ ID NO: 23, a HCDR2 as set forth in SEQ ID NO: 24, and a HCDR3 as set forth in SEQ ID NO: 25, and the VL comprises a light chain complementarity determining region (LCDR) 1 as set forth in SEQ ID NO: 28, a LCDR2 as set forth in SEQ ID NO: 29, and a LCDR3 as set forth in SEQ ID NO: 30;

[0153] (2) the VH comprises HCDR1 as set forth in SEQ ID NO: 69, HCDR2 as set forth in SEQ ID NO: 70, and HCDR3 as set forth in SEQ ID NO: 71, and the VL comprises LCDR1 as set forth in SEQ ID NO: 74, LCDR2 as set forth in SEQ ID NO: 248, and LCDR3 as set forth in SEQ ID NO: 76;

[0154] (3) the VH comprises HCDR1 as shown in SEQ ID NO: 69, HCDR2 as shown in SEQ ID NO: 70, and HCDR3 as shown in SEQ ID NO: 71, and the VL comprises LCDR1 as shown in SEQ ID NO: 74, LCDR2 as shown in SEQ ID NO: 75, and LCDR3 as shown in SEQ ID NO: 76;

[0155] (4) the VH comprises HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 5, and the VL comprises LCDR1 as shown in SEQ ID NO: 8, LCDR2 as shown in SEQ ID NO: 9, and LCDR3 as shown in SEQ ID NO: 10;

[0156] (5) the VH comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15, and the VL comprises LCDR1 as shown in SEQ ID NO: 18, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20;

[0157] (6) the VH comprises HCDR1 as shown in SEQ ID NO: 33, HCDR2 as shown in SEQ ID NO: 34, and HCDR3 as shown in SEQ ID NO: 35, and the VL comprises LCDR1 as shown in SEQ ID NO: 38, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20;

[0158] (7) the VH comprises HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43, and the VL comprises LCDR1 as shown in SEQ ID NO:46, LCDR2 as shown in SEQ ID NO:47, and LCDR3 as shown in SEQ ID NO:48;

[0159] (8) the VH comprises HCDR1 as shown in SEQ ID NO: 51, HCDR2 as shown in SEQ ID NO: 52, and HCDR3 as shown in SEQ ID NO: 53, and the VL comprises LCDR1 as shown in SEQ ID NO: 56, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20;

[0160] (9) the VH comprises HCDR1 as shown in SEQ ID NO: 59, HCDR2 as shown in SEQ ID NO: 60, and HCDR3 as shown in SEQ ID NO: 61, and the VL comprises LCDR1 as shown in SEQ ID NO: 64, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 66;

[0161] (10) the VH comprises HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 79, and the VL comprises LCDR1 as shown in SEQ ID NO: 82, LCDR2 as shown in SEQ ID NO: 83, and LCDR3 as shown in SEQ ID NO: 84;

[0162] (11) the VH comprises HCDR1 as shown in SEQ ID NO: 87, HCDR2 as shown in SEQ ID NO: 88, and HCDR3 as shown in SEQ ID NO: 89, and the VL comprises LCDR1 as shown in SEQ ID NO: 92, LCDR2 as shown in SEQ ID NO: 93, and LCDR3 as shown in SEQ ID NO: 94;

[0163] (12) the VH comprises HCDR1 as shown in SEQ ID NO: 130, HCDR2 as shown in SEQ ID NO: 131, and HCDR3 as shown in SEQ ID NO: 279, and the VL comprises LCDR1 as shown in SEQ ID NO: 74, LCDR2 as shown in SEQ ID NO: 134, and LCDR3 as shown in SEQ ID NO: 76;

[0164] (13) the VH comprises HCDR1 as shown in SEQ ID NO: 59, HCDR2 as shown in SEQ ID NO: 137, and HCDR3 as shown in SEQ ID NO: 138, and the VL comprises LCDR1 as shown in SEQ ID NO: 141, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 66;

[0165] (14) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 144, and the VL comprises LCDR1 as shown in SEQ ID NO: 147, LCDR2 as shown in SEQ ID NO: 148, and LCDR3 as shown in SEQ ID NO: 149;

[0166] (15) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 123, and the VL comprises LCDR1 as shown in SEQ ID NO: 218, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 127;

[0167] (16) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 123, and the VL comprises LCDR1 as shown in SEQ ID NO: 126, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 127;

[0168] (17) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 103, and LCDR3 as shown in SEQ ID NO: 104;

[0169] (18) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 235, and LCDR3 as shown in SEQ ID NO: 104;

[0170] (19) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 238, and LCDR3 as shown in SEQ ID NO: 104;

[0171] (20) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 232, LCDR2 as shown in SEQ ID NO: 245, and LCDR3 as shown in SEQ ID NO: 104;

[0172] (21) the VH comprises HCDR1 as shown in SEQ ID NO: 97, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 102, LCDR2 as shown in SEQ ID NO: 103, and LCDR3 as shown in SEQ ID NO: 104;

[0173] (22) the VH comprises HCDR1 as shown in SEQ ID NO: 107, HCDR2 as shown in SEQ ID NO: 108, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 102, LCDR2 as shown in SEQ ID NO: 103, and LCDR3 as shown in SEQ ID NO: 111; or

[0174] (23) The VH comprises HCDR1 as shown in SEQ ID NO: 114, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 115, and the VL comprises LCDR1 as shown in SEQ ID NO: 102, LCDR2 as shown in SEQ ID NO: 118, and LCDR3 as shown in SEQ ID NO: 104.

[0175] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention bind to pyroE Aβ3-42 peptide. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention bind to Aβ1-42 protofibril filaments.

[0176] In some embodiments, the VH and VL of the antibody each comprise the following amino acid sequence:

[0177] (1) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 164 and SEQ ID NO: 219, respectively;

[0178] (2) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 166 and SEQ ID NO: 219, respectively;

[0179] (3) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 174 and SEQ ID NO: 219, respectively;

[0180] (4) the VH and VL comprise amino acid sequences that are 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% identical to SEQ ID NO: 176 and SEQ ID NO: 219, respectively;

[0181] (5) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 21 and SEQ ID NO: 26, respectively;

[0182] (6) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 197 and SEQ ID NO: 246, respectively;

[0183] (7) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 197 and SEQ ID NO: 249, respectively;

[0184] (8) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 67 and SEQ ID NO: 72, respectively;

[0185] (9) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 1 and SEQ ID NO: 6, respectively;

[0186] (10) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 11 and SEQ ID NO: 16, respectively;

[0187] (11) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 31 and SEQ ID NO: 36, respectively;

[0188] (12) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 39 and SEQ ID NO: 44, respectively;

[0189] (13) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 49 and SEQ ID NO: 54, respectively;

[0190] (14) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 57 and SEQ ID NO: 62, respectively;

[0191] (15) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 77 and SEQ ID NO: 80, respectively;

[0192] (16) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 85 and SEQ ID NO: 90, respectively;

[0193] (17) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 128 and SEQ ID NO: 132, respectively;

[0194] (18) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 135 and SEQ ID NO: 139, respectively;

[0195] (19) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 142 and SEQ ID NO: 145, respectively;

[0196] (20) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 160 and SEQ ID NO: 216, respectively;

[0197] (21) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 152 and SEQ ID NO: 216, respectively;

[0198] (22) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 156 and SEQ ID NO: 216, respectively;

[0199] (23) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 158 and SEQ ID NO: 216, respectively;

[0200] (24) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 162 and SEQ ID NO: 216, respectively;

[0201] (25) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 160 and SEQ ID NO: 214, respectively;

[0202] (26) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 152 and SEQ ID NO: 214, respectively;

[0203] (27) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 162 and SEQ ID NO: 214, respectively;

[0204] (28) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 119 and SEQ ID NO: 124, respectively;

[0205] (29) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 230, respectively;

[0206] (30) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 233, respectively;

[0207] (31) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 236, respectively;

[0208] (32) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 185 and SEQ ID NO: 239, respectively;

[0209] (33) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 241, respectively;

[0210] (34) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 243, respectively;

[0211] (35) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 95 and SEQ ID NO: 100, respectively;

[0212] (36) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 105 and SEQ ID NO: 109, respectively; or

[0213] (37) The VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 112 and SEQ ID NO: 116, respectively.

[0214] In some embodiments, the VH and VL of the anti-Aβ antibody include functional variants formed by inserting, deleting and / or substituting one or more amino acids therein compared to the VH and VL of a reference antibody (e.g., an anti-Aβ antibody comprising the VH and VL set forth herein, e.g., an antibody comprising the VH set forth in SEQ ID NO: 164 and the VL set forth in SEQ ID NO: 219), provided that the functional variant retains the ability to bind to Aβ (e.g., Aβ1-42 protofibrils and / or pyroE Aβ3-42 peptide).

[0215] A functional variant comprises or consists of an amino acid sequence 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%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the reference antibody.

[0216] In the context of functional variants, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40% of the total number of amino acids in the reference antibody amino acid sequence, more preferably does not exceed 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0217] In some embodiments, insertions, deletions and / or substitutions may be made in the framework (FR) regions, eg, in FR1, FR2, FR3 and / or FR4.

[0218] In some embodiments, insertions, deletions and / or substitutions may be made in the CDR regions, e.g., CDR1, CDR2 and / or CDR3, provided that the insertions, deletions and / or substitutions do not affect or do not substantially affect the ability of the antibody or antigen-binding fragment thereof to bind to Aβ (e.g., Aβ1-42 protofibrils and / or pyroE Aβ3-42 peptides).

[0219] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions include substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or substitution of one charged or polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, etc. Preferred exemplary conservative substitutions include the following changes: alanine to serine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glycine to proline; histidine to asparagine or glutamine; lysine to arginine, glutamine or glutamic acid; phenylalanine to tyrosine, serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; etc.

[0220] In some preferred embodiments, the VH of the anti-Aβ antibody comprises the amino acid sequence shown in SEQ ID NO: 164, and the VL comprises the amino acid sequence shown in SEQ ID NO: 219. In some preferred embodiments, the VH of the anti-Aβ antibody comprises the amino acid sequence shown in SEQ ID NO: 160, and the VL comprises the amino acid sequence shown in SEQ ID NO: 216.

[0221] Based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of the antibody can be divided into lambda (λ) chain and kappa (κ) chain. The antibodies disclosed herein can be any of the above classes or subtypes.

[0222] In some embodiments, the antibody is an antibody isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is an antibody subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0223] In some embodiments, the antibodies of the present invention comprise an Fc region. The Fc region can be of any isotype or subtype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can comprise one or more substitutions or modifications. In some embodiments, the Fc region is an IgG1 Fc region or is derived therefrom, optionally comprising one or more substitutions or modifications. In some embodiments, the Fc region comprises one or more amino acid substitutions that extend the half-life of the antibody. In preferred embodiments, the Fc region comprises the L234A and L235A amino acid substitutions.

[0224] In some embodiments, the anti-Aβ antibodies of the invention comprise a heavy chain (HC) and a light chain (LC), wherein:

[0225] (1) the HC and LC comprise amino acid sequences that have 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 to SEQ ID NO: 275 and SEQ ID NO: 277, respectively; or

[0226] (2) the HC and LC comprise amino acid sequences 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 to SEQ ID NO: 271 and SEQ ID NO: 273, respectively.

[0227] In some embodiments, the HC and LC of the anti-Aβ antibody include functional variants formed by inserting, deleting and / or substituting one or more amino acids therein compared to the HC and LC of a reference antibody (e.g., an antibody comprising a HC with an amino acid sequence as shown in SEQ ID NO: 275 and a LC with an amino acid sequence as shown in SEQ ID NO: 277, or an antibody comprising a HC with an amino acid sequence as shown in SEQ ID NO: 271 and a LC with an amino acid sequence as shown in SEQ ID NO: 273), provided that the functional variant retains the ability to bind to Aβ (e.g., Aβ1-42 protofibrils and / or pyroE Aβ3-42 peptide).

[0228] A functional variant comprises or consists of an amino acid sequence 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%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the reference antibody.

[0229] In some embodiments, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the reference antibody amino acid sequence, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0230] In some embodiments, insertions, deletions and / or substitutions may be made in the framework (FR) regions, e.g., FR1, FR2, FR3 and / or FR4; and / or constant regions, e.g., CL, CH1, CH2 and / or CH3.

[0231] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0232] In some embodiments, the antibodies of the present invention are selected from murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0233] The antibodies of the present invention may be intact antibodies or antigen-binding fragments thereof. The antigen-binding fragment may be any fragment of an antibody that retains the ability to bind to Aβ. Examples of antigen-binding fragments include, but are not limited to, full-length antibodies; Fab; F(ab')2; Fab'; Fd; Fd'; Fv; scFv; dAb; individual complementarity determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fds (VH-CH1-VH-CH1), and modified forms of any of the foregoing fragments that retain antigen-binding activity.

[0234] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv), and ds-scFv.

[0235] In some embodiments, the antibody is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

[0236] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0237] Nucleic acid molecules

[0238] In yet another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present invention.

[0239] For example, the present invention provides nucleic acid molecules encoding the CDR sequences in any one of the VH and / or VL disclosed herein. In some embodiments, the present invention also provides nucleic acid molecules having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid molecules encoding the CDR sequences in any one of the VH and / or VL disclosed herein.

[0240] For another example, the present invention provides nucleic acid molecules encoding any one of the VH and / or VL disclosed herein. In some embodiments, the present invention also provides nucleic acid molecules encoding any one of the VH and / or VL disclosed herein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0241] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 165; and / or, a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 220.

[0242] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 161; and / or, a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 217.

[0243] For another example, the present invention provides nucleic acid molecules encoding any one of the heavy and / or light chains of the antibodies disclosed herein. In some embodiments, the present invention also provides nucleic acid molecules encoding any one of the heavy and / or light chains of the antibodies disclosed herein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0244] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 276; and / or, a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 278.

[0245] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 272; and / or, a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 274.

[0246] In a more preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 276 and the nucleotide sequence shown in SEQ ID NO: 278. In another more preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 272 and the nucleotide sequence shown in SEQ ID NO: 274.

[0247] carrier

[0248] In another aspect, the present invention provides a vector comprising a nucleic acid molecule disclosed herein, which encodes an anti-Aβ antibody or antigen-binding fragment thereof disclosed herein.

[0249] In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising the heavy chain or light chain variable region of an antibody.For example, the present invention provides an expression vector comprising any of the above-mentioned nucleic acid molecules.

[0250] Any vector can be suitable for use in the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV) vector, a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pBY, pGAR, pBABE-puro, pBABE-neo largeT cDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0251] The vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, vectors can be selected from pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript sequence (Stratagene, LaJolla, Calif.), pET sequence (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors that can be used for the present invention include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the present invention include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0252] Recombinant expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain a replication system functional in prokaryotic or eukaryotic host cells. The replication system can be derived, for example, from ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0253] For example, the vector can be an adenovirus vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector can be administered to a subject, and then enters the subject's cells in vivo, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cells, and then the cells express the antibody disclosed herein.

[0254] cell

[0255] In another aspect, the present invention provides a cell comprising a nucleic acid molecule disclosed herein or a vector disclosed herein.

[0256] Any cell can be used as the host cell of the nucleic acid molecule of the present invention or vector. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include but are not limited to true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (Enterobactehaceae), such as Escherichia (Escherichia), such as Escherichia coli (E.coli); Enterobacter (Enterobacter); Erwinia (Erwinia); Klebsiella (Klebsiella); Proteus (Proteus); Salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); Serratia (Serratia), such as Serratia marcescens (Serratia marcescens). marcescans and Shigella; Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0257] The cells of the present invention are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The cells of the present invention can be administered to a subject, and the cells express the antibodies disclosed herein in vivo.

[0258] Preparation method

[0259] In another aspect, the present invention provides a method for preparing the antibodies or antigen-binding fragments thereof disclosed herein, comprising culturing the cells provided by the present invention under conditions suitable for expression of the antibodies or antigen-binding fragments thereof disclosed herein; and isolating the antibodies or antigen-binding fragments thereof from the culture and / or culture supernatant of the cells.

[0260] Any method suitable for producing antibodies can be used to produce the antibodies of the present invention. For example, antibodies can be prepared using a hybridoma method, such as the method described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0261] Immunizing agents generally include protein antigens, fragments thereof, or fusion proteins thereof. In this context, any suitable form of Aβ can be used as an immunizing agent (antigen) to generate non-human antibodies specific for Aβ and screen for the biological activity of the antibodies. For example, antigens suitable for use in the present invention include, but are not limited to, Aβ1-42 protofibrils, KLH-CV-41-Aβ1-40, Aβ1-40 protofibrils, and pyroE Aβ3-42 peptides.

[0262] The immunizing agent can be used alone or in combination with one or more immunogenicity enhancers known in the art. Typically, if human cells are needed, peripheral blood lymphocytes are used; if non-human mammalian sources are needed, spleen cells or lymph node cells are used. Lymphocytes are then fused with immortalized cell lines using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically will contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0263] Then can determine whether there is monoclonal antibody for antigen in the culture medium of culture hybridoma cell.Preferably, the binding specificity of the monoclonal antibody produced by hybridoma cell is measured by immunoprecipitation or by in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such technology and determination are known in the art.The binding affinity of monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107:220 (1980).In addition, in the therapeutic application of monoclonal antibody, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

[0264] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites of a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0265] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). The hybridoma cells of the present invention are used as a preferred source of such DNA. The isolated DNA can be placed in an expression vector and then transfected into a host cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain antibodies in recombinant host cells that synthesize monoclonal antibodies. The DNA can also be modified, for example, by replacing the homologous mouse sequence with the coding sequence of the human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0266] The sequences of the antibodies of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0267] Once the relevant sequence is obtained, the relevant antibody can be obtained in large quantities using recombinant methods. This typically involves cloning the sequence into a vector, transferring it into cells, and then isolating the relevant antibody from the propagated host cells using conventional methods. Alternatively, synthetic methods can be used to synthesize the relevant sequence, especially when the fragment length is relatively short. Typically, very long fragments of sequence can be obtained by first synthesizing multiple small fragments and then ligating them. The nucleic acid molecule can then be introduced into various existing vectors known in the art, which can be used to transform appropriate host cells to enable protein expression, thereby obtaining the antibodies of the present invention.

[0268] Pharmaceutical composition

[0269] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, or a cell disclosed herein, and optionally a pharmaceutically acceptable carrier.

[0270] The antibodies or antigen-binding fragments thereof of the present invention and derivatives, fragments, analogs and homologs thereof can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise an antibody or antigen-binding fragment thereof or an agent and a pharmaceutically acceptable carrier.

[0271] As used herein, the term "pharmaceutically acceptable" refers to a carrier or excipient that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0272] The exemplary carriers used in pharmaceutical compositions of the present invention include saline, buffered saline, glucose and water. The exemplary excipients used in compositions of the present invention include fillers, adhesives, disintegrants, coating agents, adsorbents, antiadherents, glidants, preservatives, antioxidants, flavorings, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators. Suitable excipients are known to those skilled in the art to prepare pharmaceutical compositions of the present invention. Usually, the selection of suitable excipients especially depends on the desired dosage form of employed activating agent, disease to be treated and compositions.

[0273] Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media for pharmaceutically active substances is well known in the art. Unless any conventional media is incompatible with the active compound, it is contemplated for use in the composition. Supplementary active compounds may also be incorporated into the composition.

[0274] The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended route of administration. The pharmaceutical compositions of the present invention are preferably administered parenterally, by injection, or orally. Injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The pharmaceutical compositions are in various dosage forms conventional in the art, preferably in solid, semisolid, or liquid form, i.e., aqueous, non-aqueous, or suspension solutions, more preferably tablets, capsules, granules, injections, or infusions. More preferably, they are administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the pharmaceutical compositions can also be administered as an aerosol or coarse spray, i.e., nasally; or intrathecally, intramedullary, or intraventricularly. More preferably, the pharmaceutical compositions can also be administered transdermally, percutaneously, topically, enterally, intravaginally, sublingually, or rectally. The pharmaceutical compositions of the present invention can be formulated into various dosage forms as needed, and a physician can determine the dosage that is beneficial to the patient based on factors such as the patient's type, age, weight, general condition, and route of administration. Administration may be by injection or other therapeutic methods.

[0275] The dosage level of the pharmaceutical composition of the present invention can be adjusted according to the amount of the composition reaching the required diagnosis or treatment result. The administration regimen can also be a single injection or multiple injections, or adjusted. The selected dosage level and regimen depend on the activity and stability (that is, half-life) of the pharmaceutical composition, preparation, route of administration, combination with other drugs or treatment, disease to be detected and / or treated or illness and the health status of the subject to be treated and various factors such as previous medical history and reasonably adjust.

[0276] Reagent test kit

[0277] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein, and instructions for administering the above-mentioned substances. In some embodiments, the kit further comprises an administration device.

[0278] In some embodiments, the present invention provides a test kit, which may include an antibody disclosed herein or its antigen-binding fragment, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein. It may include an antibody, nucleic acid molecule, vector, cell, or pharmaceutical composition of the present invention in a single common container. In some cases, the test kit may include instructions for use, which include information about the antibody, nucleic acid molecule, vector, cell, or pharmaceutical composition in the test kit and the dosage form. Typically, such information helps patients and physicians to effectively and safely use the encapsulated antibody, nucleic acid molecule, vector, cell, or pharmaceutical composition. The container used in such a test kit may typically include at least one vial, test tube, flask, bottle, syringe, or other suitable container.

[0279] In some embodiments, the present invention provides a kit, which may include an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein, instructions for administering the above-mentioned substance, and an administration device for administering the above-mentioned substance. The administration device can introduce a substance into the patient's body through a parenteral route (e.g., intramuscularly, subcutaneously, or intravenously). For example, the administration device can be a syringe (e.g., a syringe pre-filled with an antibody or pharmaceutical composition of the present invention, such as an automatic syringe), which can include a syringe and a needle (which can be used to pierce the skin and / or blood vessels) for accommodating a fluid to be injected (e.g., an antibody or pharmaceutical composition of the present invention). The mode of administration can be changed. The route of administration can include oral administration, intramuscular injection, subcutaneous injection, rectal administration, etc.

[0280] Methods and uses

[0281] In another aspect, the present invention provides a method for treating or preventing a disease caused by abnormal accumulation or deposition of Aβ in a subject, which comprises administering to the subject an effective amount of the antibody or antigen-binding fragment thereof disclosed herein, the nucleic acid molecule disclosed herein, the vector disclosed herein, the cell disclosed herein, or the pharmaceutical composition disclosed herein.

[0282] In another aspect, the present invention provides a method for clearing or alleviating abnormal accumulation or deposition of Aβ in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof disclosed herein, the nucleic acid molecule disclosed herein, the vector disclosed herein, the cell disclosed herein, or the pharmaceutical composition disclosed herein.

[0283] As used herein, the term "effective amount" means an amount of a compound that, when administered to a subject for the treatment or prevention of a disease, is sufficient to achieve the therapeutic or preventive effects described herein. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for preventive treatment.

[0284] In some embodiments, the dosage or effective amount administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount to administer to a human or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the antibody and the route of administration.

[0285] The dosage of the antibodies, nucleic acid molecules, vectors, cells or pharmaceutical compositions described herein can be administered to a mammal at once or in a series of sub-doses over an appropriate time period, for example, daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, bi-monthly or annually as needed. A dosage unit comprising an effective amount of an antibody, nucleic acid molecule, vector, cell and / or pharmaceutical composition can be administered as a single daily dose, or the total daily dose can be administered as two, three, four or more divided doses administered daily as needed.

[0286] The appropriate mode of administration can be selected by the physician. The route of administration can be parenteral administration, for example, by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. The dosage and method of administration can vary according to the weight, age, condition, etc. of the subject and can be appropriately selected.

[0287] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof disclosed herein, the nucleic acid molecule disclosed herein, the vector disclosed herein, the cell disclosed herein, or the pharmaceutical composition disclosed herein in the preparation of a medicament for treating or preventing a disease caused by abnormal accumulation or deposition of Aβ in a subject.

[0288] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof disclosed herein, the nucleic acid molecule disclosed herein, the vector disclosed herein, the cell disclosed herein, or the pharmaceutical composition disclosed herein in the preparation of a medicament for clearing or alleviating abnormal accumulation or deposition of Aβ in a subject.

[0289] In some embodiments, the subject treated with the methods described herein has a central nervous system degenerative disease. In some embodiments, the subject has Alzheimer's disease (AD), Parkinson's disease (PD), Down syndrome or cerebral amyloid angiopathy, or is at risk of developing AD, PD, Down syndrome or cerebral amyloid angiopathy. In some embodiments, the subject has preclinical AD or clinical AD, e.g., prodromal AD, mild AD, moderate AD or severe AD.

[0290] In the case of Alzheimer's disease, "treating" or "preventing" can limit, slow down or stop one or more of the patient's cognitive symptoms, behavioral symptoms and / or pathological symptoms, or prevent the onset or progression of one or more of the above symptoms. Cognitive symptoms can include decreased intelligence, difficulty thinking and understanding, confusion in the evening, delusions, disorientation, forgetfulness, fabricated stories, mental confusion, difficulty concentrating, inability to form new memories, inability to do simple math, or inability to recognize common objects. Behavioral symptoms can include aggressiveness, restlessness, difficulty with self-care, irritability, meaningless monologues, personality changes, restlessness, lack of restraint, or wandering and getting lost. AD patients may also experience anger, dullness, general dissatisfaction, loneliness, mood swings, depression, hallucinations, paranoia, uncoordinated muscle movements, slurred speech or loss of appetite. Pathological symptoms include the formation and development of amyloid plaques and neurofibrillary tangles.

[0291] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described below in conjunction with specific examples and accompanying drawings, and the advantages and features of the present invention will become clearer as the description progresses. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are carried out according to conventional conditions in the art, for example, the conditions described in Sambrook and Russeii et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press, or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available. Example

[0292] Example 1. Production of anti-Aβ antibodies

[0293] The antigens used in the present invention were synthesized by Gill Biochemical.

[0294] Use 10mM NaOH solution, add it to Aβ1-42 powder, vortex for 2 minutes, until Aβ is completely dissolved (concentration is 443uM), add PBS and shake, place at 37℃ for 48 hours, take the supernatant after centrifugation, use Superdex 75 column to purify the original fine fibrils, package and place in a -80℃ refrigerator for immunization and screening.

[0295] The pyroE Aβ3-42 peptide antigen was dissolved in 50 mM sodium acetate for use in immunization and screening.

[0296] 1) Mouse immunization

[0297] Six Balb / c mice, purchased from Zhejiang Weitong Lihua, were immunized four times with Aβ1-42 protofibrils and pyroE Aβ3-42 peptide antigens. The first immunization was with complete Freund's adjuvant (CFA). Two weeks later, immunizations were repeated weekly using a Sigma Adjuvant System. Group C1 received 79 μL of Aβ1-42 protofibril antigen mixed with 421 μL of PBS, while group C2 received 500 μL of pyroE Aβ3-42 peptide antigen. A 3 mL syringe was used to draw 500 μL of the prepared antigen mixture, and another syringe was filled with 500 μL of CFA. The mixture was mixed using a T-junction. A water-in-oil emulsion was formed. A drop was then placed in ice water and held for 20 minutes to prevent diffusion. The prepared antigen was administered subcutaneously to the abdomen near the groin, at a dose of 50 μg / 120 μL per mouse. 100 μL of each was injected subcutaneously into the abdomen near the groin. After subcutaneous immunization, mice were briefly anesthetized with isoflurane and immunized by footpad injection at 20 μL per mouse. Immunized mice were returned to their cages and continued to be housed. Groups C1 and C2 received their first immunization with complete Freund's adjuvant, with a 2-week interval between the first and second immunizations.

[0298] Starting with the second immunization, groups C1 and C2 were immunized with Ribi adjuvant once a week for three weeks. For group C1, 24 μL of antigen was mixed with 176 μL of PBS; for group C2, 150 μL of antigen was mixed with 50 μL of PBS. The diluted antigen was added to 200 μL of the Sigma Adjuvant System and vortexed thoroughly for 2 minutes to mix thoroughly. After briefly anesthetizing the mice with isoflurane, the hind paws or elbows of the mice were disinfected with alcohol cotton pads. A disposable insulin syringe was used to inject 25 μL of the antigen-adjuvant mixture into each hind paw or elbow. Immunized mice were returned to their cages and, after waking and showing no abnormalities, were returned to their cages for continued care. After immunization, serum titers were measured. If fusion requirements were met, spleen cell fusion and subsequent hybridoma screening were initiated.

[0299] 2) Spleen cell fusion

[0300] After the test titer reaches the antibody screening standard, SP2 / 0 cells are revived. The first batch of fused SP2 / 0 cells was donated, and the second and third batches of fused SP2 / 0 cells were purchased from Qiwen Company. Open the water bath and heat it to 37°C in advance. In the clean bench, aseptically remove 30mL of Medium A (STEMCELL, 03801) and place it in a 50mL centrifuge tube. Place it in the water bath to preheat. Open the liquid nitrogen tank, remove the SP2 / 0 cells, and quickly transfer them to a 37°C water bath and shake them to dissolve within 2 minutes. Wipe off the water on the outer wall of the cryopreservation tube, spray it with 75% ethanol, and place it in the clean bench. Unscrew the cryopreservation tube cap, gently blow it twice with a pipette, and then remove all the cells into a 15mL centrifuge tube. Then add the preheated Medium A (STEMCELL, 03801) drop by drop, for a total of 5mL. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and pat the cell pellet to disperse. Resuspend in 10 mL of Medium A and count the cells. Inoculate the cells into a T75 culture flask at a density of 5e4 cells / mL in a total volume of 20 mL. Culture the flask in a 37°C 5% CO2 incubator.

[0301] Cells should be passaged when they reach an appropriate density. The maximum cell density per T75 flask at each passage is 4e5 cells / mL. The day before electrofusion, seed a T175 flask at a density of 1e5 cells / mL. The optimal density at electrofusion the next day is 3-4e5 cells / mL.

[0302] Eyeballs were enucleated for blood collection, which was then placed in EP tubes (heparinized), incubated at 37°C for 2 h, and then centrifuged to separate serum. Serum was then stored at -20°C until use (positive control). Mice were sacrificed by cervical dislocation, disinfected by immersion in 75% alcohol solution for 5 min, and then fixed with the abdomen facing up on a dissecting board in a clean bench. Popliteal, inguinal, and iliac lymph nodes were removed from the mice and repeatedly ground to collect as many lymphocytes as possible, which were then filtered and counted.

[0303] SP2 / 0 cells and B cells were mixed in a specific ratio and resuspended in electrofusion buffer. The cell suspension was immediately added to the electrodes, and after setting the parameters, fusion was performed. After electrofusion, the cells were allowed to rest for at least 15 minutes. The cells were gently removed from the electrofusion cuvette and cultured overnight. The cells were then added to Medium C (STEMCELL, 03803) and Medium D (STEMCELL, 03804). The semi-solid culture medium was seeded at a density of 2 mL / well in a 6-well plate and cultured. Fluorescent clones were selected for subsequent screening after the cells reached an appropriate level.

[0304] 3) Antibody screening

[0305] Dilute Aβ1-42 protofibrils or pyroE Aβ3-42 peptide in coating solution, add to the ELISA plate, and incubate overnight at 4°C. Remove the plate, wash three times with PBST, add blocking solution to the plate, and incubate at room temperature for 1 hour. Wash once with PBST, add 50 μL / well of the sample and control to the plate, and incubate at room temperature for 1 hour. Wash the plate with PBST, dilute the secondary antibody, add to the plate, and incubate at room temperature for 1 hour. Wash the plate again with TBST, add the colorimetric solution, and incubate in a 37°C incubator for 10 minutes. Add 2M concentrated sulfuric acid stop solution and read the plate within 15 minutes using a microplate reader (wavelength: 450 nm).

[0306] Example 2. Acquisition of anti-Aβ antibody hybridoma positive clones

[0307] Based on the results of hybridoma screening, positive single clones were centrifuged at 1000 rpm, harvested, and total RNA extracted with Trizol. This RNA was used as a template for first-strand cDNA synthesis. The variable region DNA sequence corresponding to the hybridoma was subsequently amplified using the first-strand cDNA as a template. To a 50 μL reaction system, 1 μL of cDNA, 5 μL of 10× PCR buffer, 1 μL each of forward and reverse primers, 1 μL of dNTPs, 1 μL of 25 mmol MgCl₂, 39 μL of H₂O, and 1 μL of Taq enzyme were added. The mixture was pre-denatured at 95°C for 10 minutes and then subjected to temperature cycling for PCR amplification. The reaction conditions were 30 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 15 seconds, followed by a 10-minute incubation at 72°C. The obtained clones such as 7C8 bound to Aβ1-42 protofibrils or pyroE Aβ3-42 peptide, and weakly bound to or did not bind to Aβ1-42 monomers. The binding activities of each chimeric antibody to the antigen are shown in Table 1.

[0308] Table 1 Binding of anti-Aβ chimeric antibodies to antigens

[0309] “++++” represents strong binding, “++” represents weak binding, “+” represents very weak binding, and “-” represents no binding.

[0310] The variable region sequences of the above-mentioned chimeric antibodies are shown in Table 2 below.

[0311] Table 2 Variable region sequences of anti-Aβ chimeric antibodies

[0312] In the amino acid sequences of VH or VL shown below, the CDR sequences are shown in bold and underlined.

[0313] 192E15

[0314] The VH amino acid sequence is shown in SEQ ID NO.1, its encoding nucleic acid is shown in SEQ ID NO.2, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.3, 4 and 5, respectively.

[0315] Nucleic acid sequence

[0316] The VL amino acid sequence is shown in SEQ ID NO.6, the encoding nucleic acid is shown in SEQ ID NO.7, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.8, 9 and 10, respectively.

[0317] Nucleic acid sequence

[0318] 179M22

[0319] The VH amino acid sequence is shown in SEQ ID NO.11, its encoding nucleic acid is shown in SEQ ID NO.12, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.13, 14 and 15, respectively.

[0320] Nucleic acid sequence

[0321] The VL amino acid sequence is shown in SEQ ID NO. 16, the encoding nucleic acid is shown in SEQ ID NO. 17, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 18, 19 and 20, respectively.

[0322] Nucleic acid sequence

[0323] 20001

[0324] The VH amino acid sequence is shown in SEQ ID NO. 21, its encoding nucleic acid is shown in SEQ ID NO. 22, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 23, 24 and 25, respectively.

[0325] Nucleic acid sequence

[0326] The VL amino acid sequence is shown in SEQ ID NO. 26, its encoding nucleic acid is shown in SEQ ID NO. 27, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 28, 29 and 30, respectively.

[0327] Nucleic acid sequence

[0328] 175P19

[0329] The VH amino acid sequence is shown in SEQ ID NO.31, its encoding nucleic acid is shown in SEQ ID NO.32, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.33, 34 and 35, respectively.

[0330] Nucleic acid sequence

[0331] The VL amino acid sequence is shown in SEQ ID NO. 36, its encoding nucleic acid is shown in SEQ ID NO. 37, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 38, 19 and 20, respectively.

[0332] Nucleic acid sequence

[0333] 178D4

[0334] The VH amino acid sequence is shown in SEQ ID NO.39, its encoding nucleic acid is shown in SEQ ID NO.40, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.41, 42 and 43, respectively.

[0335] Nucleic acid sequence

[0336] The VL amino acid sequence is shown in SEQ ID NO.44, its encoding nucleic acid is shown in SEQ ID NO.45, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.46, 47 and 48, respectively.

[0337] Nucleic acid sequence

[0338] 185O18

[0339] The VH amino acid sequence is shown in SEQ ID NO.49, its encoding nucleic acid is shown in SEQ ID NO.50, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.51, 52 and 53, respectively.

[0340] Nucleic acid sequence

[0341] The VL amino acid sequence is shown in SEQ ID NO. 54, its encoding nucleic acid is shown in SEQ ID NO. 55, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 56, 19 and 20, respectively.

[0342] Nucleic acid sequence

[0343] 193D22

[0344] The VH amino acid sequence is shown in SEQ ID NO.57, its encoding nucleic acid is shown in SEQ ID NO.58, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.59, 60 and 61, respectively.

[0345] Nucleic acid sequence

[0346] The VL amino acid sequence is shown in SEQ ID NO.62, its encoding nucleic acid is shown in SEQ ID NO.63, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.64, 65 and 66, respectively.

[0347] Nucleic acid sequence

[0348] 151N5

[0349] The VH amino acid sequence is shown in SEQ ID NO.67, its encoding nucleic acid is shown in SEQ ID NO.68, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.69, 70 and 71, respectively.

[0350] Nucleic acid sequence

[0351] The VL amino acid sequence is shown in SEQ ID NO.72, its encoding nucleic acid is shown in SEQ ID NO.73, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.74, 75 and 76, respectively.

[0352] Nucleic acid sequence

[0353] 156N21

[0354] The VH amino acid sequence is shown in SEQ ID NO. 77, its encoding nucleic acid is shown in SEQ ID NO. 78, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 3, 4 and 79, respectively.

[0355] Nucleic acid sequence

[0356] The VL amino acid sequence is shown in SEQ ID NO.80, its encoding nucleic acid is shown in SEQ ID NO.81, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.82, 83 and 84, respectively.

[0357] Nucleic acid sequence

[0358] 171P1

[0359] The VH amino acid sequence is shown in SEQ ID NO.85, its encoding nucleic acid is shown in SEQ ID NO.86, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.87, 88 and 89, respectively.

[0360] Nucleic acid sequence

[0361] The VL amino acid sequence is shown in SEQ ID NO.90, the encoding nucleic acid is shown in SEQ ID NO.91, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.92, 93 and 94, respectively.

[0362] Nucleic acid sequence

[0363] 6B5

[0364] The VH amino acid sequence is shown in SEQ ID NO.95, its encoding nucleic acid is shown in SEQ ID NO.96, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.97, 98 and 99, respectively.

[0365] Nucleic acid sequence

[0366] The VL amino acid sequence is shown in SEQ ID NO.100, the encoding nucleic acid is shown in SEQ ID NO.101, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.102, 103 and 104, respectively.

[0367] Nucleic acid sequence

[0368] 6C9

[0369] The VH amino acid sequence is shown in SEQ ID NO.105, its encoding nucleic acid is shown in SEQ ID NO.106, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.107, 108 and 99, respectively.

[0370] Nucleic acid sequence

[0371] The VL amino acid sequence is shown in SEQ ID NO.109, the encoding nucleic acid is shown in SEQ ID NO.110, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.102, 103 and 111, respectively.

[0372] Nucleic acid sequence

[0373] 11G10

[0374] The VH amino acid sequence is shown in SEQ ID NO.112, its encoding nucleic acid is shown in SEQ ID NO.113, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.114, 98 and 115, respectively.

[0375] Nucleic acid sequence

[0376] The VL amino acid sequence is shown in SEQ ID NO.116, the encoding nucleic acid is shown in SEQ ID NO.117, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.102, 118 and 104, respectively.

[0377] Nucleic acid sequence

[0378] 7C8

[0379] The VH amino acid sequence is shown in SEQ ID NO.119, its encoding nucleic acid is shown in SEQ ID NO.120, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.121, 122 and 123, respectively.

[0380] Nucleic acid sequence

[0381] The VL amino acid sequence is shown in SEQ ID NO. 124, the encoding nucleic acid is shown in SEQ ID NO. 125, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 126, 65 and 127, respectively.

[0382] Nucleic acid sequence

[0383] 174J17

[0384] The VH amino acid sequence is shown in SEQ ID NO.128, its encoding nucleic acid is shown in SEQ ID NO.129, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.130, 131 and 279, respectively.

[0385] Nucleic acid sequence

[0386] The VL amino acid sequence is shown in SEQ ID NO.132, its encoding nucleic acid is shown in SEQ ID NO.133, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.74, 134 and 76, respectively.

[0387] Nucleic acid sequence

[0388] 193M7

[0389] The VH amino acid sequence is shown in SEQ ID NO.135, its encoding nucleic acid is shown in SEQ ID NO.136, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.59, 137 and 138, respectively.

[0390] Nucleic acid sequence

[0391] The VL amino acid sequence is shown in SEQ ID NO.139, its encoding nucleic acid is shown in SEQ ID NO.140, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.141, 65 and 66, respectively.

[0392] Nucleic acid sequence

[0393] 15E11-2

[0394] The VH amino acid sequence is shown in SEQ ID NO.142, its encoding nucleic acid is shown in SEQ ID NO.143, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.121, 122 and 144, respectively.

[0395] Nucleic acid sequence

[0396] The VL amino acid sequence is shown in SEQ ID NO. 145, the encoding nucleic acid is shown in SEQ ID NO. 146, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 147, 148 and 149, respectively.

[0397] Nucleic acid sequence

[0398] Example 3. Evaluation of phagocytic activity of anti-Aβ chimeric antibodies

[0399] There are microglia in the brain, which mediate the endogenous immune response to central nervous system damage and disease, thereby exerting neuroprotective or neurotoxic effects. Microglia are responsible for clearing dead cell debris, dendritic spines, extracellular matrix and invading pathogens through phagocytosis. Microglia in AD patients can clear Aβ through phagocytosis. Antibodies can be incubated with 5XFAD mouse brain slices in vitro. After the antibodies bind to Aβ in the brain slices, BV2 microglia are added, and the Fc of the antibody binds to the FcR of BV2, thereby mediating the phagocytosis of BV2. The present invention uses this method to evaluate the effect of antibodies in promoting the phagocytosis of Aβ.

[0400] The chimeric antibody samples were diluted to 100 μg / mL and 10 μg / mL with culture medium for later use. The pre-cut 10 μm FAD mouse brain slices were taken out and the sample loading range was circled with a histochemical pen. 400 μL of the prepared sample to be tested was gently added to the slices and incubated at 37°C for 1 hour. During this period, mouse microglial cells BV2 were prepared and resuspended in culture medium to 4×10 5 / mL, discard the original sample on the slice after antibody incubation, add 500μL cell suspension, culture for 24h, change the medium once, and continue to culture for another 24h, for a total of 48h.

[0401] Remove the slices after phagocytosis, collect the supernatant of the slices and centrifuge, and collect the brain tissue on the slices with 75 μL of guanidine hydrochloride lysis buffer. Collect twice to obtain a total of 150 μL lysis buffer sample. After thorough mixing, continue lysis on ice for 1-2 hours. Mix several times during this period. After lysis is completed, the sample can be stored at -80°C or immediately subjected to ELISA quantitative detection.

[0402] Aβ1-42 in samples was quantified using the Human Aβ42 ELISA Kit (Cat: KHB3441, Invitrogen). Samples were centrifuged at 3000-4000 rpm, and 20 μL of the supernatant was diluted 1000-fold. The assay was performed according to the kit instructions, and the Aβ1-42 concentration in the sample was back-calculated using the standard curve. The phagocytosis results of different chimeric antibody samples are shown in Figure 1. Clones 151N5, 200O1, 6B5, and 7C8 all promoted Aβ phagocytosis by microglia. Clones 200O1 and 192E15 exhibited phagocytic activity comparable to that of Donanemab, while clones 7C8 and 7C8 exhibited phagocytic activity comparable to that of Lecanemab, demonstrating that these chimeric antibodies have excellent potential for clearing Aβ plaques.

[0403] Example 4. Binding activity of anti-Aβ chimeric antibodies to antigens

[0404] Using biomembrane interferometry (BLI / Gator Prime), anti-Aβ chimeric antibodies were immobilized with a ProA probe. Aβ1-42 protofibrils were serially diluted with HBS-EP + buffer (Cytiva, BR10069) at three concentration points to test the affinity of anti-Aβ chimeric antibodies 7C8 and 6B5 for the antigen. Furthermore, the pyroE Aβ3-16-biotin peptide was immobilized on the SA probe surface. Anti-Aβ chimeric antibodies 200O1 and 151N5, serially diluted with HBS-EP + buffer, were used as analytes to test the binding of the pyroE Aβ3-16-biotin peptide to the chimeric antibodies. The results are shown in Figure 2. Anti-Aβ chimeric antibodies 151N5 and 200O1 bind strongly to pyroE Aβ3-16, while anti-Aβ chimeric antibodies 6B5 and 7C8 bind more strongly to protofibrils.

[0405] Example 5. Construction of anti-Aβ humanized antibodies

[0406] 1) Design of humanized antibodies

[0407] Combining the results of mouse brain slice phagocytic activity detection and the developability evaluation of chimeric antibodies, four chimeric antibodies 7C8, 151N5, 200O1, and 6B5 were humanized.

[0408] The chimeric antibody 7C8 sequence was aligned with human germline antibody sequences to identify highly homologous human germline light chain genes (IMGT_hVK2-28 and IGKJ2*01) and human germline heavy chain genes (IMGT_hVH2-5 or IMGT_hVH4-59 and IGHJ4*01) that maintain the core hydrophobic structure of the antibody and are highly recurrent in humans. Mouse antibody CDRs were then transplanted. Computer-generated homology modeling was performed to analyze the CDR regions and surrounding framework amino acid sequences to avoid concentrated distribution of surface charge or hydrophobic regions. Seven heavy chain variants, h7C8-H1 to H7, and two light chain variants, h7C8-K1 to K2, were designed.

[0409] Similarly, the chimeric antibody 151N5 sequence was aligned with the human antibody germline sequence to determine the human germline light chain genes IMGT_hVK1-27, IGKJ2*01 and the human germline heavy chain genes IMGT_hVH3-21, IGHJ4*01, and mouse antibody CDR transplantation was performed; a total of 6 heavy chain variants h151N5-H1~H6 and 9 light chain variants h151N5-K1~K9 were designed.

[0410] Similarly, the chimeric antibody 200O1 sequence was aligned with the human antibody germline sequence to determine the human germline light chain genes IMGT_hVK1-27, IGKJ2*01 and the human germline heavy chain genes IMGT_hVH2-26 or IMGT_hVH4-59, IGHJ4*01, and mouse antibody CDR transplantation was performed; a total of 9 heavy chain variants h200O1-H1~H9 and 4 light chain variants h200O1-K1~K4 were designed.

[0411] Similarly, the chimeric antibody 6B5 sequence was aligned with the human antibody germline sequence to determine the human germline light chain genes IMGT_hVK3-20 or IMGT_hVK1-39, IGKJ2*01 and the human germline heavy chain genes IMGT_hVH1-46, IGHJ4*01, and mouse antibody CDR transplantation was performed; a total of 9 heavy chain variants h6B5-H1~H5 and 4 light chain variants h6B5-K1~K6 were designed.

[0412] The heavy and light chain variable region sequences of the above-mentioned anti-Aβ humanized antibodies are shown in Tables 3 and 4, respectively.

[0413] Table 3. Heavy chain variable region sequences of anti-Aβ humanized antibodies

[0414] h7C8 H1

[0415] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.150, the encoding nucleic acid is shown in SEQ ID NO.151, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.121, 122 and 123, respectively.

[0416] Nucleic acid sequence

[0417] h7C8 H2

[0418] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.152, the encoding nucleic acid is shown in SEQ ID NO.153, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.121, 122 and 123, respectively.

[0419] Nucleic acid sequence

[0420] h7C8 H3

[0421] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.154, the encoding nucleic acid is shown in SEQ ID NO.155, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.121, 122 and 123, respectively.

[0422] Nucleic acid sequence

[0423] h7C8 H4

[0424] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.156, the encoding nucleic acid is shown in SEQ ID NO.157, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.121, 122 and 123, respectively.

[0425] Nucleic acid sequence

[0426] h7C8 H5

[0427] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.158, the encoding nucleic acid is shown in SEQ ID NO.159, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.121, 122 and 123, respectively.

[0428] Nucleic acid sequence

[0429] h7C8 H6

[0430] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 160, the encoding nucleic acid is shown in SEQ ID NO. 161, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 121, 122 and 123, respectively.

[0431] Nucleic acid sequence

[0432] h7C8 H7

[0433] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.162, the encoding nucleic acid is shown in SEQ ID NO.163, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.121, 122 and 123, respectively.

[0434] Nucleic acid sequence

[0435] h200O1 H1

[0436] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.164, its encoding nucleic acid is shown in SEQ ID NO.165, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.23, 24 and 25, respectively.

[0437] Nucleic acid sequence

[0438] h200O1 H2

[0439] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.166, its encoding nucleic acid is shown in SEQ ID NO.167, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.23, 24 and 25, respectively.

[0440] Nucleic acid sequence

[0441] h200O1 H3

[0442] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.168, its encoding nucleic acid is shown in SEQ ID NO.169, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.23, 170 and 25, respectively.

[0443] Nucleic acid sequence

[0444] h200O1 H4

[0445] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 171, its encoding nucleic acid is shown in SEQ ID NO. 172, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 23, 173 and 25, respectively.

[0446] Nucleic acid sequence

[0447] h200O1 H5

[0448] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 174, the encoding nucleic acid is shown in SEQ ID NO. 175, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 23, 24 and 25, respectively.

[0449] Nucleic acid sequence

[0450] h200O1 H6

[0451] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.176, its encoding nucleic acid is shown in SEQ ID NO.177, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.23, 24 and 25, respectively.

[0452] Nucleic acid sequence

[0453] h200O1 H7

[0454] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 178, the encoding nucleic acid is shown in SEQ ID NO. 179, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 23, 170 and 25, respectively.

[0455] Nucleic acid sequence

[0456] h200O1 H8

[0457] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 180, the encoding nucleic acid is shown in SEQ ID NO. 181, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 23, 173 and 25, respectively.

[0458] Nucleic acid sequence

[0459] h200O1 H9

[0460] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 182, its encoding nucleic acid is shown in SEQ ID NO. 183, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 23, 184 and 25, respectively.

[0461] Nucleic acid sequence

[0462] h6B5 H1

[0463] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.185, the encoding nucleic acid is shown in SEQ ID NO.186, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.97, 98 and 99, respectively.

[0464] Nucleic acid sequence

[0465] h6B5 H2

[0466] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.187, the encoding nucleic acid is shown in SEQ ID NO.188, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.97, 98 and 189, respectively.

[0467] Nucleic acid sequence

[0468] h6B5 H3

[0469] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 190, the encoding nucleic acid is shown in SEQ ID NO. 191, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 192, 98 and 189, respectively.

[0470] Nucleic acid sequence

[0471] h6B5 H4

[0472] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 193, the encoding nucleic acid is shown in SEQ ID NO. 194, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 192, 98 and 189, respectively.

[0473] Nucleic acid sequence

[0474] h6B5 H5

[0475] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 195, the encoding nucleic acid is shown in SEQ ID NO. 196, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 192, 98 and 189, respectively.

[0476] Nucleic acid sequence

[0477] h151N5 H1

[0478] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.197, the encoding nucleic acid is shown in SEQ ID NO.198, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.69, 70 and 71, respectively.

[0479] Nucleic acid sequence

[0480] h151N5 H2

[0481] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.199, its encoding nucleic acid is shown in SEQ ID NO.200, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.201, 202 and 71, respectively.

[0482] Nucleic acid sequence

[0483] h151N5 H3

[0484] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 203, its encoding nucleic acid is shown in SEQ ID NO. 204, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 201, 205 and 71, respectively.

[0485] Nucleic acid sequence

[0486] h151N5 H4

[0487] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 206, its encoding nucleic acid is shown in SEQ ID NO. 207, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 201, 205 and 279, respectively.

[0488] Nucleic acid sequence

[0489] h151N5 H5

[0490] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.208, its encoding nucleic acid is shown in SEQ ID NO.209, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.201, 205 and 210, respectively.

[0491] Nucleic acid sequence

[0492] h151N5 H6

[0493] The amino acid sequence of the heavy chain VH is shown in SEQ ID NO. 211, its encoding nucleic acid is shown in SEQ ID NO. 212, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 201, 213 and 210, respectively.

[0494] Nucleic acid sequence

[0495] Table 4. Light chain variable region sequences of anti-Aβ humanized antibodies

[0496] h7C8 K1

[0497] The amino acid sequence of the light chain VK is shown in SEQ ID NO.214, its encoding nucleic acid is shown in SEQ ID NO.215, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.126, 65 and 127, respectively.

[0498] Nucleic acid sequence

[0499] h7C8 K2

[0500] The amino acid sequence of the light chain VK is shown in SEQ ID NO.216, its encoding nucleic acid is shown in SEQ ID NO.217, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.218, 65 and 127, respectively.

[0501] Nucleic acid sequence

[0502] h200O1 K1

[0503] The amino acid sequence of the light chain VK is shown in SEQ ID NO.219, its encoding nucleic acid is shown in SEQ ID NO.220, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.28, 29 and 30, respectively.

[0504] Nucleic acid sequence

[0505] h200O1 K2

[0506] The amino acid sequence of the light chain VK is shown in SEQ ID NO.221, its encoding nucleic acid is shown in SEQ ID NO.222, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.223, 29 and 30, respectively.

[0507] Nucleic acid sequence

[0508] h200O1 K3

[0509] The amino acid sequence of the light chain VK is shown in SEQ ID NO. 224, the encoding nucleic acid is shown in SEQ ID NO. 225, and the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 226, 29 and 30, respectively.

[0510] Nucleic acid sequence

[0511] h200O1 K4

[0512] The amino acid sequence of the light chain VK is shown in SEQ ID NO.227, its encoding nucleic acid is shown in SEQ ID NO.228, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.226, 229 and 30, respectively.

[0513] Nucleic acid sequence

[0514] h6B5 K1

[0515] The amino acid sequence of the light chain VK is shown in SEQ ID NO.230, its encoding nucleic acid is shown in SEQ ID NO.231, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.232, 103 and 104, respectively.

[0516] Nucleic acid sequence

[0517] h6B5 K2

[0518] The amino acid sequence of the light chain VK is shown in SEQ ID NO.233, its encoding nucleic acid is shown in SEQ ID NO.234, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.232, 235 and 104, respectively.

[0519] Nucleic acid sequence

[0520] h6B5 K3

[0521] The amino acid sequence of the light chain VK is shown in SEQ ID NO.236, its encoding nucleic acid is shown in SEQ ID NO.237, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.232, 238 and 104, respectively.

[0522] Nucleic acid sequence

[0523] h6B5 K4

[0524] The amino acid sequence of the light chain VK is shown in SEQ ID NO.239, its encoding nucleic acid is shown in SEQ ID NO.240, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.232, 103 and 104, respectively.

[0525] Nucleic acid sequence

[0526] h6B5 K5

[0527] The amino acid sequence of the light chain VK is shown in SEQ ID NO.241, its encoding nucleic acid is shown in SEQ ID NO.242, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.232, 103 and 104, respectively.

[0528] Nucleic acid sequence

[0529] h6B5 K6

[0530] The amino acid sequence of the light chain VK is shown in SEQ ID NO.243, its encoding nucleic acid is shown in SEQ ID NO.244, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.232, 245 and 104, respectively.

[0531] Nucleic acid sequence

[0532] h151N5 K1

[0533] The amino acid sequence of the light chain VK is shown in SEQ ID NO.246, its encoding nucleic acid is shown in SEQ ID NO.247, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.74, 248 and 76, respectively.

[0534] Nucleic acid sequence

[0535] h151N5 K2

[0536] The amino acid sequence of the light chain VK is shown in SEQ ID NO.249, its encoding nucleic acid is shown in SEQ ID NO.250, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.74, 248 and 76, respectively.

[0537] Nucleic acid sequence

[0538] h151N5 K3

[0539] The amino acid sequence of the light chain VK is shown in SEQ ID NO.251, its encoding nucleic acid is shown in SEQ ID NO.252, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.74, 253 and 76, respectively.

[0540] Nucleic acid sequence

[0541] h151N5 K4

[0542] The amino acid sequence of the light chain VK is shown in SEQ ID NO.254, its encoding nucleic acid is shown in SEQ ID NO.255, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.256, 257 and 76, respectively.

[0543] Nucleic acid sequence

[0544] h151N5 K5

[0545] The amino acid sequence of the light chain VK is shown in SEQ ID NO.258, its encoding nucleic acid is shown in SEQ ID NO.259, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.260, 261 and 76, respectively.

[0546] Nucleic acid sequence

[0547] h151N5 K6

[0548] The amino acid sequence of the light chain VK is shown in SEQ ID NO.262, its encoding nucleic acid is shown in SEQ ID NO.263, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.260, 261 and 264, respectively.

[0549] Nucleic acid sequence

[0550] h151N5 K7

[0551] The amino acid sequence of the light chain VK is shown in SEQ ID NO.265, its encoding nucleic acid is shown in SEQ ID NO.266, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.74, 75 and 76, respectively.

[0552] Nucleic acid sequence

[0553] h151N5 K8

[0554] The amino acid sequence of the light chain VK is shown in SEQ ID NO.267, its encoding nucleic acid is shown in SEQ ID NO.268, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.74, 75 and 76, respectively.

[0555] Nucleic acid sequence

[0556] h151N5 K9

[0557] The amino acid sequence of the light chain VK is shown in SEQ ID NO.269, its encoding nucleic acid is shown in SEQ ID NO.270, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs.74, 75 and 76, respectively.

[0558] Nucleic acid sequence

[0559] 2) Construction of humanized antibodies

[0560] The variable region sequence of the light chain was synthesized and cloned into a eukaryotic expression plasmid containing the antibody kappa chain constant region Ckappa. Simultaneously, the variable region sequence of the heavy chain was synthesized and cloned into a eukaryotic expression plasmid containing the human IgG1 constant region CH1-CH3. The above light and heavy chain plasmids were combined and paired, and then transfected into CHO-S cells. The cells were expressed at 37°C for 5-6 days, and the culture supernatant was collected and purified through a Protein A column to obtain a humanized bivalent antibody.

[0561] Example 6. Affinity detection of anti-Aβ humanized antibodies

[0562] Using surface plasmon resonance (SPR / Biacore T200), Aβ1-42 protofibrils were immobilized on the surface of a CM5 chip via amino-coupling. A serially diluted 6B5 / 7C8 humanized antibody was then passed over the chip as the analyte, and the real-time binding signal between the antibody and the antigen was detected. The affinities of the 6B5 / 7C8 humanized antibody are shown in Figure 3 and Table 5. Using surface plasmon resonance (SPR / Biacore T200), pyroE Aβ3-16-biotin and Aβ3-16-biotin antigens were coupled to different channels of an SA chip, respectively. A serially diluted 200O1 / 151N5 humanized antibody was then passed over the chip as the analyte, and the real-time binding signal between the antibody and the antigen was detected. The affinities of the antibodies are shown in Figure 4 and Table 6.

[0563] Table 5. Affinity of 7C8 and 6B5 humanized antibodies

[0564] Table 6. Affinity of 200O1 and 151N5 humanized antibodies

[0565] Example 7. Phagocytic activity of humanized antibodies

[0566] After the initial screening, the humanized antibody samples with good phagocytic activity were diluted to 100 μg / mL and 10 μg / mL with culture medium for use. The pre-cut 10 μm FAD mouse brain slices were taken out and the sample addition range was circled with a histochemical pen. 400 μL of the prepared sample to be tested was gently added to the slices and incubated at 37°C for 1 hour. During this period, mouse microglial cells BV2 were prepared and resuspended in culture medium to 4×10 5 / mL, discard the original sample on the slice after antibody incubation, add 500μL cell suspension, culture for 24h, change the medium once, and continue to culture for another 24h, for a total of 48h.

[0567] Remove the slices after phagocytosis, collect the supernatant of the slices and centrifuge, and collect the brain tissue on the slices with 75 μL of guanidine hydrochloride lysis buffer. Collect twice to obtain a total of 150 μL lysis buffer sample. After thorough mixing, continue lysis on ice for 1-2 hours. Mix several times during this period. After lysis is completed, the sample can be stored at -80°C or immediately subjected to ELISA quantitative detection.

[0568] Aβ1-42 in samples was quantified using the Human Aβ42 ELISA Kit (Cat: KHB3441, Invitrogen). Samples were centrifuged at 3000-4000 rpm, and 20 μL of the supernatant was diluted 1000-fold. The assay was performed according to the kit instructions, and the Aβ1-42 concentration in the sample was back-calculated based on the standard curve. The phagocytosis results of the humanized samples are shown in Figure 5. Both 200O1 H1K1 and 7C8 H6K2 promote Aβ phagocytosis in BV2 microglial cells.

[0569] Example 8. Construction of long-acting humanized anti-Aβ antibody

[0570] Alzheimer's disease patients require long-term medication. To enhance the convenience and reduce the dosage of antibodies, the development of long-acting anti-Aβ antibodies holds great promise. In the Phase I clinical trial of lecanemab, the antibody level in the 1 mg / kg group quickly fell below the detection limit, making it impossible to calculate the half-life. The half-life of the 10 mg / kg group was 6.9 days. In the Phase I / II clinical trial of donanemab, the half-life of the intravenous 1 mg / kg group was 3.19 days, and the half-life of the 10 mg / kg group was 10.5 days. Both half-lives are shorter than those of typical monoclonal antibodies. Therefore, monoclonal antibodies with longer half-lives may achieve better efficacy. Based on the 7C8 H6K2 and 200O1 H1K1 sequences, their sequences were optimized to increase the half-life of the antibodies. The sequences of the constructed long-acting antibodies are shown in Table 7.

[0571] Table 7. Sequences of long-acting humanized anti-Aβ antibodies

[0572] The heavy chain amino acid sequence of h7C8 H6K2 hIgG1 LA is shown in SEQ ID NO. 271, and the encoding nucleic acid is shown in SEQ ID NO. 272.

[0573] Nucleic acid sequence

[0574] The light chain amino acid sequence of h7C8 H6K2 hIgG1 LA is shown in SEQ ID NO. 273, and the encoding nucleic acid is shown in SEQ ID NO. 274.

[0575] Nucleic acid sequence

[0576] The heavy chain amino acid sequence of h200O1 H1K1 hIgG1 LA is shown in SEQ ID NO. 275, and the encoding nucleic acid is shown in SEQ ID NO. 276.

[0577] Nucleic acid sequence

[0578] The light chain amino acid sequence of h200O1 H1K1 hIgG1 LA is shown in SEQ ID NO. 277, and the encoding nucleic acid is shown in SEQ ID NO. 278.

[0579] Nucleic acid sequence

[0580] Example 9. Half-life detection of long-acting humanized anti-Aβ antibodies

[0581] Thirty female B-FcRn mice, 6 to 8 weeks old and weighing 18 to 20 g (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), were selected and randomly divided into 6 groups of 5 mice each. The dose was set at 10 mg / kg, a single dose of 100 μL / mouse, and administration was performed by tail vein injection. The dosing and grouping are shown in Table 8. Blood was collected from the submandibular vein of the mice at 0 h, 1 h, 4 h, D1, D3, D5, D7, D14, D21, and D28 of administration. 7 to 8 drops of fresh blood were collected from the mice, incubated at 37°C for 30 min, and then centrifuged at 4500 rpm for 15 min to collect serum. The serum concentration of the antibody was determined by ELISA. The dosing concentration was calculated based on the average weight of B-FcRn mice of 19 g. The specific dosing groups and doses are shown in Table 8 below.

[0582] Plot the curve by subtracting the blank well value from the standard and sample values. If replicate wells are used, calculate the average value. Plot the curve using the serum drug concentration as the y-axis and the blood collection time as the x-axis. If the sample is diluted before testing, multiply the final calculation by the dilution factor to obtain the actual sample concentration.

[0583] As shown in Figure 6, the half-life of the h7C8 H6K2 antibody molecule is basically equivalent to that of Lecanemab, but weaker than that of h7C8 H6K2 LA. The half-life of the h200O1 H1K1 antibody molecule is basically equivalent to that of Donanemab, but weaker than that of h200O1 H1K1 LA.

[0584] Table 8. Dosage groups and dosages

[0585] Example 10. Phagocytic activity detection of long-acting humanized anti-Aβ antibodies

[0586] The long-acting antibody sample was diluted to 100 μg / mL and 10 μg / mL with culture medium for later use. The pre-cut 10 μm FAD mouse brain slices were taken out and the sample addition range was circled with a histochemical pen. 400 μL of the prepared sample to be tested was gently added to the slices and incubated at 37°C for 1 hour. During this period, BV2 cells (mouse microglia) were prepared and resuspended in culture medium to 4×10 5 / mL, discard the original sample on the slice after antibody incubation, add 500μL cell suspension, culture for 24h, change the medium once, and continue to culture for another 24h, for a total of 48h.

[0587] Remove the slices after phagocytosis, collect the supernatant of the slices and centrifuge, and collect the brain tissue on the slices with 75 μL of guanidine hydrochloride lysis buffer. Collect twice to obtain a total of 150 μL lysis buffer sample. After thorough mixing, continue lysis on ice for 1-2 hours. Mix several times during this period. After lysis is completed, the sample can be stored at -80°C or immediately subjected to ELISA quantitative detection.

[0588] Aβ1-42 was quantified using the Human Aβ42 ELISA Kit (Cat: KHB3441, Invitrogen). Samples were centrifuged at 3000-4000 rpm, and 20 μL of the supernatant was diluted 1000-fold. The assay was performed according to the kit instructions, and the Aβ1-42 concentration in the sample was back-calculated based on the standard curve. The results of phagocytosis by long-acting antibodies are shown in Figure 7. Both 200O1 H1K1 LA and 7C8 H6K2 LA effectively cleared Aβ from brain slices.

[0589] Example 11. Immunogenicity testing of long-acting antibodies

[0590] In this study, peripheral venous blood was collected from several healthy volunteers, PBMCs were isolated, and cells were resuspended in X-vivo15 medium (Lonza, 04-418Q) at 2.5 × 10 5 PBMCs were incubated with 100 μg / mL CM383 monoclonal antibody for 48 hours, and CD4 + CD134 in T cells + (OX40 + ) and CD137 + (4-1BB + ) The percentage of double-positive cells was determined using the same method, using keyhole limpet hemocyanin (KLH) as a positive control. The results (see Figure 8 and Table 9) showed that the immunogenicity of 200O1 H1K1 LA and 7C8 H6K2 LA was lower than that of donanemab and lecanemab.

[0591] Table 9. Percentage of CD134+ (OX40+) and CD137+ (4-1BB+) double-positive cells in CD4+ T cells

[0592] Example 12. Analysis of the binding activity of humanized antibodies and long-acting antibodies to negative screening cells

[0593] The HEK293-APP stably transfected cell line expressing amyloid precursor protein APP was used as the target cell. The cell density was adjusted to 1×10 cells per 1 mL using the prepared BSA blocking solution. 7 Cells were incubated on ice for 30 minutes. During this time, antibody samples were prepared at a concentration of 50 μg / mL and diluted 3-fold. After blocking, flow cytometry buffer was added to dilute the cells to a density of 5×10 cells per 1 mL. 5 100 μL of the antibody sample was inoculated into a U-shaped 96-well plate, and the supernatant was discarded after centrifugation. 100 μL of the serially diluted antibody sample was added to each well and incubated with the cells at 4°C for 60 minutes.

[0594] After incubation, the cells were washed once with 200 μL of flow cytometry buffer. 50 μL of AF647 goat anti-human IgG and Fcγ secondary antibody diluent (Jackson ImmunoResearch, 109-606-170, 1:300 dilution) was added to each well to label the cell surface antibodies. After incubation on ice for 20 minutes, 50 μL of PI solution (Sigma, P4170, 1:100 dilution) was added and incubated for another 5 minutes. After washing once, the cells were resuspended in 70 μL of PBS and the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry. Antibody concentration-MFI curves were obtained using a four-parameter model. The results are shown in Figure 9 and Table 10. h200O1 H1K1 LA did not bind to AMP, demonstrating excellent binding specificity. h7C8 H6K2 LA weakly bound to AMP, but the binding strength was significantly weaker than that of Lecanemab, indicating that h7C8 H6K2 LA had better binding specificity.

[0595] Table 10. Binding of different antibodies to APP

[0596] NA indicates that the antibody has weak binding ability to APP or does not bind to APP, and EC cannot be fitted. 50 result

[0597] Example 13. Detection of brain concentration of long-acting antibodies

[0598] Twelve C57BL / 6 mice, 12 months old and weighing 25-30 g (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), were randomly divided into four groups of three mice each. h7C8 H6K2 LA, h200O1 H1K1 LA, lecanemab, and donanemab were biotinylated and administered, and antibody concentrations in mouse brain tissue were measured using ELISA. A single dose of 30 mg / kg was administered in a volume of 100 μL per mouse via tail vein injection. Seven days after administration, the mouse brains were harvested, homogenized, centrifuged, and the supernatant was collected for ELISA analysis of antibody concentrations in brain tissue. As shown in Figure 10 and Table 11, after seven days of administration, the brain concentration of the 7C8 humanized antibody variant h7C8 H6K2 LA tended to be higher than that of lecanemab, while the brain concentration of the 200O1 humanized antibody variant h200O1 H1K1 LA was significantly higher than that of donanemab.

[0599] Table 11. Concentrations of different long-acting antibodies in brain tissue

[0600] Example 14. Effect of humanized antibodies on the Aβ plaque area in the hippocampus and striatum of mice

[0601] Twenty-five 6-week-old 5X FAD transgenic mice weighing approximately 20 g (Jiangsu Jinzhihe Biotechnology Co., Ltd.) were randomly divided into five groups of five mice each. The drug was administered at a dose of 10 mg / kg twice weekly for three months. To prevent the development of anti-drug antibodies against the humanized antibody during long-term administration, the Fc residue of the test drug was modified to mIgG2a in this example.

[0602] After administration, the rat brains were harvested and frozen sections were prepared from half. Immunohistochemical staining of the cortex, cerebellum, and corpus callosum for Aβ was performed. Insoluble Aβ was extracted from the other half of the brain using guanidine hydrochloride, and Aβ1-42 was quantified using the Human Aβ42 ELISA Kit (Cat: KHB3441, Invitrogen). As shown in Figure 11 and Table 12, 7C8 mIgG2a and 200O1 mIgG2a showed a more pronounced reduction in insoluble Aβ than lecanemab and donanemab, significantly reducing Aβ plaque area in the hippocampus and striatum.

[0603] Table 12. Average Aβ plaque areas in the hippocampus and striatum of 5X FAD mice after administration of different antibodies

[0604] Example 15. Effect of humanized antibodies on soluble and insoluble Aβ levels in mouse brain

[0605] 5X FAD mice

[0606] Six-week-old 5X FAD transgenic mice (Jiangsu Jinzhihe Biotechnology Co., Ltd.) were selected and divided into a 7C8 mIgG2a low-dose group (3 mg / kg), a 7C8 mIgG2a medium-dose group (10 mg / kg), a 7C8 mIgG2a high-dose group (30 mg / kg), and an anti-KLH mIgG2a group (30 mg / kg). The animals were dosed twice a week for 13 weeks, for a total of 27 doses. A blank control was also set up. At 13 weeks, the right hemispheres of the animals were collected for brain tissue soluble Aβ1-42 and insoluble Aβ1-42, as well as brain plaque staining. The percentage reduction of soluble and insoluble Aβ was calculated relative to the anti-KLH mIgG2a group. The results are shown in Figure 12 and Table 13. For 5X FAD mice, 7C8 mIgG2a significantly reduced the content of soluble and insoluble Aβ in the brain.

[0607] Table 13. Percentage reduction of soluble and insoluble Aβ in brain tissue of 5X FAD mice after administration of humanized antibodies

[0608] 6-week-old FAD 4T mice

[0609] Six-week-old FAD4T transgenic mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were selected and divided into a low-dose 7C8 mIgG2a group (3 mg / kg), a medium-dose 7C8 mIgG2a group (10 mg / kg), a high-dose 7C8 mIgG2a group (30 mg / kg), and an anti-KLH mIgG2a group (30 mg / kg). Animals were dosed twice weekly for 13 weeks, for a total of 27 doses. At week 13, the right hemispheres of the animals were harvested for brain tissue analysis of soluble and insoluble Aβ1-42, as well as plaque staining. The percentage reduction in soluble and insoluble Aβ was calculated relative to the anti-KLH mIgG2a group. As shown in Figure 13 and Table 14, 7C8 mIgG2a significantly reduced the levels of soluble and insoluble Aβ in the brains of six-week-old FAD4T mice.

[0610] Table 14. Percentage reduction of soluble and insoluble Aβ in brain tissue of FAD 4T mice (6 weeks old) after administration of humanized antibodies

[0611] 15-week-old FAD 4T mice

[0612] Fifteen-week-old FAD4T transgenic mice (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were selected and divided into a low-dose 7C8 mIgG2a group (3 mg / kg), a medium-dose 7C8 mIgG2a group (10 mg / kg), a high-dose 7C8 mIgG2a group (30 mg / kg), and an anti-KLH mIgG2a group (30 mg / kg). Animals were dosed twice weekly for 13 weeks, for a total of 27 doses. At week 13, the right hemispheres of the animals were harvested for brain tissue analysis of soluble and insoluble Aβ1-42, as well as plaque staining. The percentage reduction of soluble and insoluble Aβ was calculated relative to the anti-KLH mIgG2a group. The results, as shown in Figure 14 and Table 15, show that 7C8 mIgG2a was able to reduce both soluble and insoluble Aβ levels in the brain of 15-week-old FAD4T mice.

[0613] Table 15. Percentage reduction of soluble and insoluble Aβ in brain tissue of FAD 4T mice (15 weeks old) after administration of humanized antibodies

Claims

1. An antibody or an antigen-binding fragment thereof that binds to β-amyloid protein (β-Amyloid, Aβ), the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises a heavy chain complementary determining region (HCDR) 1 as shown in SEQ ID NO: 23, a HCDR2 as shown in SEQ ID NO: 24, and a HCDR3 as shown in SEQ ID NO: 25, and the VL comprises a light chain complementary determining region (LCDR) 1 as shown in SEQ ID NO: 28, a LCDR2 as shown in SEQ ID NO: 29, and a LCDR3 as shown in SEQ ID NO: 30; (2) the VH comprises HCDR1 as shown in SEQ ID NO:69, HCDR2 as shown in SEQ ID NO:70, and HCDR3 as shown in SEQ ID NO:71, and the VL comprises LCDR1 as shown in SEQ ID NO:74, LCDR2 as shown in SEQ ID NO:248, and LCDR3 as shown in SEQ ID NO:76; (3) the VH comprises HCDR1 as shown in SEQ ID NO:69, HCDR2 as shown in SEQ ID NO:70, and HCDR3 as shown in SEQ ID NO:71, and the VL comprises LCDR1 as shown in SEQ ID NO:74, LCDR2 as shown in SEQ ID NO:75, and LCDR3 as shown in SEQ ID NO:76; (4) the VH comprises HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, and HCDR3 as shown in SEQ ID NO:5, and the VL comprises LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:10; (5) the VH comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15, and the VL comprises LCDR1 as shown in SEQ ID NO: 18, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20; (6) the VH comprises HCDR1 as shown in SEQ ID NO:33, HCDR2 as shown in SEQ ID NO:34, and HCDR3 as shown in SEQ ID NO:35, and the VL comprises LCDR1 as shown in SEQ ID NO:38, LCDR2 as shown in SEQ ID NO:19, and LCDR3 as shown in SEQ ID NO:20; (7) the VH comprises HCDR1 as shown in SEQ ID NO:41, HCDR2 as shown in SEQ ID NO:42, and HCDR3 as shown in SEQ ID NO:43, and the VL comprises LCDR1 as shown in SEQ ID NO:46, LCDR2 as shown in SEQ ID NO:47, and LCDR3 as shown in SEQ ID NO:48; (8) the VH comprises HCDR1 as shown in SEQ ID NO:51, HCDR2 as shown in SEQ ID NO:52, and HCDR3 as shown in SEQ ID NO:53, and the VL comprises LCDR1 as shown in SEQ ID NO:56, LCDR2 as shown in SEQ ID NO:19, and LCDR3 as shown in SEQ ID NO:20; (9) the VH comprises HCDR1 as shown in SEQ ID NO:59, HCDR2 as shown in SEQ ID NO:60, and HCDR3 as shown in SEQ ID NO:61, and the VL comprises LCDR1 as shown in SEQ ID NO:64, LCDR2 as shown in SEQ ID NO:65, and LCDR3 as shown in SEQ ID NO:66; (10) the VH comprises HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, and HCDR3 as shown in SEQ ID NO:79, and the VL comprises LCDR1 as shown in SEQ ID NO:82, LCDR2 as shown in SEQ ID NO:83, and LCDR3 as shown in SEQ ID NO:84; (11) the VH comprises HCDR1 as shown in SEQ ID NO:87, HCDR2 as shown in SEQ ID NO:88, and HCDR3 as shown in SEQ ID NO:89, and the VL comprises LCDR1 as shown in SEQ ID NO:92, LCDR2 as shown in SEQ ID NO:93, and LCDR3 as shown in SEQ ID NO:94; (12) the VH comprises HCDR1 as shown in SEQ ID NO: 130, HCDR2 as shown in SEQ ID NO: 131, and HCDR3 as shown in SEQ ID NO: 279, and the VL comprises LCDR1 as shown in SEQ ID NO: 74, LCDR2 as shown in SEQ ID NO: 134, and LCDR3 as shown in SEQ ID NO: 76; (13) the VH comprises HCDR1 as shown in SEQ ID NO:59, HCDR2 as shown in SEQ ID NO:137, and HCDR3 as shown in SEQ ID NO:138, and the VL comprises LCDR1 as shown in SEQ ID NO:141, LCDR2 as shown in SEQ ID NO:65, and LCDR3 as shown in SEQ ID NO:66; (14) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 144, and the VL comprises LCDR1 as shown in SEQ ID NO: 147, LCDR2 as shown in SEQ ID NO: 148, and LCDR3 as shown in SEQ ID NO: 149; (15) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 123, and the VL comprises LCDR1 as shown in SEQ ID NO: 218, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO:127; (16) the VH comprises HCDR1 as shown in SEQ ID NO: 121, HCDR2 as shown in SEQ ID NO: 122, and HCDR3 as shown in SEQ ID NO: 123, and the VL comprises LCDR1 as shown in SEQ ID NO: 126, LCDR2 as shown in SEQ ID NO: 65, and LCDR3 as shown in SEQ ID NO: 127; (17) the VH comprises HCDR1 as shown in SEQ ID NO:97, HCDR2 as shown in SEQ ID NO:98, and HCDR3 as shown in SEQ ID NO:99, and the VL comprises LCDR1 as shown in SEQ ID NO:232, LCDR2 as shown in SEQ ID NO:103, and LCDR3 as shown in SEQ ID NO:104; (18) the VH comprises HCDR1 as shown in SEQ ID NO:97, HCDR2 as shown in SEQ ID NO:98, and HCDR3 as shown in SEQ ID NO:99, and the VL comprises LCDR1 as shown in SEQ ID NO:232, LCDR2 as shown in SEQ ID NO:235, and LCDR3 as shown in SEQ ID NO:104; (19) the VH comprises HCDR1 as shown in SEQ ID NO:97, HCDR2 as shown in SEQ ID NO:98, and HCDR3 as shown in SEQ ID NO:99, and the VL comprises LCDR1 as shown in SEQ ID NO:232, LCDR2 as shown in SEQ ID NO:238, and LCDR3 as shown in SEQ ID NO:104; (20) the VH comprises HCDR1 as shown in SEQ ID NO:97, HCDR2 as shown in SEQ ID NO:98, and HCDR3 as shown in SEQ ID NO:99, and the VL comprises LCDR1 as shown in SEQ ID NO:232, LCDR2 as shown in SEQ ID NO:245, and LCDR3 as shown in SEQ ID NO:104; (21) the VH comprises HCDR1 as shown in SEQ ID NO:97, HCDR2 as shown in SEQ ID NO:98, and HCDR3 as shown in SEQ ID NO:99, and the VL comprises LCDR1 as shown in SEQ ID NO:102, LCDR2 as shown in SEQ ID NO:103, and LCDR3 as shown in SEQ ID NO:104; (22) the VH comprises HCDR1 as shown in SEQ ID NO: 107, HCDR2 as shown in SEQ ID NO: 108, and HCDR3 as shown in SEQ ID NO: 99, and the VL comprises LCDR1 as shown in SEQ ID NO: 102, LCDR2 as shown in SEQ ID NO: 103, and LCDR3 as shown in SEQ ID NO: 111; or (23) the VH comprises HCDR1 as shown in SEQ ID NO: 114, HCDR2 as shown in SEQ ID NO: 98, and HCDR3 as shown in SEQ ID NO: 115, and the VL It comprises LCDR1 as shown in SEQ ID NO:102, LCDR2 as shown in SEQ ID NO:118, and LCDR3 as shown in SEQ ID NO:

104.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to the pyroE Aβ3-42 peptide.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to Aβ1-42 protofibrils.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: (1) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 164 and SEQ ID NO: 219, respectively; (2) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 166 and SEQ ID NO: 219, respectively; (3) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 174 and SEQ ID NO: 219, respectively; (4) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 176 and SEQ ID NO: 219, respectively; (5) the VH and VL comprise amino acid sequences 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 to SEQ ID NO:21 and SEQ ID NO:26, respectively; (6) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 197 and SEQ ID NO: 246, respectively; (7) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 197 and SEQ ID NO: 249, respectively; (8) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 67 and SEQ ID NO: 72, respectively; (9) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 1 and SEQ ID NO: 6, respectively; (10) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 11 and SEQ ID NO: 16, respectively; (11) the VH and VL comprise amino acid sequences 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 to SEQ ID NO:31 and SEQ ID NO:36, respectively; (12) the VH and VL comprise amino acid sequences 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 to SEQ ID NO:39 and SEQ ID NO:44, respectively; (13) the VH and VL comprise amino acid sequences 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 to SEQ ID NO:49 and SEQ ID NO:54, respectively; (14) the VH and VL comprise amino acid sequences 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 to SEQ ID NO:57 and SEQ ID NO:62, respectively; (15) the VH and VL comprise amino acid sequences 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 to SEQ ID NO:77 and SEQ ID NO:80, respectively; (16) the VH and VL comprise amino acid sequences 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 to SEQ ID NO:85 and SEQ ID NO:90, respectively; (17) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 128 and SEQ ID NO: 132, respectively; (18) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 135 and SEQ ID NO: 139, respectively; (19) The VH and VL respectively contain the same amino acids as SEQ ID NO: 142 and SEQ ID NO:

145. amino acid sequences 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; (20) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 160 and SEQ ID NO: 216, respectively; (21) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 152 and SEQ ID NO: 216, respectively; (22) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 156 and SEQ ID NO: 216, respectively; (23) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 158 and SEQ ID NO: 216, respectively; (24) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 162 and SEQ ID NO: 216, respectively; (25) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 160 and SEQ ID NO: 214, respectively; (26) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 152 and SEQ ID NO: 214, respectively; (27) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 162 and SEQ ID NO: 214, respectively; (28) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 119 and SEQ ID NO: 124, respectively; (29) The VH and VL respectively contain at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 1 amino acid sequences having at least 98%, at least 99%, or 100% sequence identity; (30) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 233, respectively; (31) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 236, respectively; (32) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 239, respectively; (33) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 241, respectively; (34) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 185 and SEQ ID NO: 243, respectively; (35) the VH and VL comprise amino acid sequences 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 to SEQ ID NO: 95 and SEQ ID NO: 100, respectively; (36) the VH and VL comprise amino acid sequences that have 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 to SEQ ID NO: 105 and SEQ ID NO: 109, respectively; or (37) The VH and VL comprise amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:112 and SEQ ID NO:116, respectively.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody is an antibody isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is of an antibody subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody comprises an IgG1 constant region (Fc region); optionally, the Fc region comprises an amino acid substitution that prolongs the half-life of the antibody, preferably, the amino acid substitution comprises L234A and L235A substitutions.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein: (1) the HC and LC comprise amino acid sequences 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 to SEQ ID NO: 275 and SEQ ID NO: 277, respectively; or (2) the HC and LC comprise amino acid sequences 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 to SEQ ID NO: 271 and SEQ ID NO: 273, respectively.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, single-chain Fv (scFv) and ds-scFv.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is a monovalent antibody, a bivalent antibody or a multivalent antibody.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

13. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A cell comprising the nucleic acid molecule of claim 13 or the vector of claim 14.

16. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the method comprising: a) culturing the cell of claim 15 under conditions suitable for expression of the antibody or antigen-binding fragment thereof; and b) isolating the antibody or antigen-binding fragment thereof from the culture medium and / or culture supernatant of the cell.

17. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14 or the cell according to claim 15, and optionally, a pharmaceutically acceptable carrier or excipient.

18. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14, the cell according to claim 15 or the pharmaceutical composition according to claim 17, and instructions for use; optionally, the kit further comprises an administration device.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14, the cell according to claim 15 or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating or preventing a disease caused by abnormal accumulation or deposition of Aβ in a subject.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid molecule according to claim 13, the vector according to claim 14, the cell according to claim 15 or the pharmaceutical composition according to claim 17 in the preparation of a medicament for clearing or alleviating abnormal accumulation or deposition of Aβ in a subject.

21. The use according to claim 19 or 20, wherein the subject suffers from a degenerative disease of the central nervous system.

22. The use of claim 21, wherein the subject suffers from, or is at risk of suffering from, Alzheimer's disease (AD), Parkinson's disease (PD), Down syndrome, or cerebral amyloid angiopathy.

23. The use of claim 22, wherein the subject suffers from preclinical AD or clinical AD, for example, prodromal AD, mild AD, moderate AD or severe AD.

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