Anti-n3pglu amyloid β antibody and use thereof

Abstract

Provided are an anti-N3pGlu amyloid β antibody and a use thereof, and a pharmaceutical composition comprising the anti-N3pGlu amyloid β antibody. The anti-N3pGlu amyloid β antibody has significantly excellent affinity, stability and specificity. Also provided is the use of the anti-N3pGlu amyloid β antibody for treating diseases caused by β amyloid (e.g., Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy).

Description

Anti-N3pGlu amyloid β antibody and its uses

[0001] Cross-reference of related applications

[0002] This application claims priority to Chinese invention patent application No. 202410926772.1, filed on July 11, 2024, entitled "Anti-N3pGlu Amyloid β Antibody and Its Use Thereof", and Chinese invention patent application No. 202510805968.X, filed on June 16, 2025, entitled "Anti-N3pGlu Amyloid β Antibody and Its Use Thereof", the contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure provides an antibody that binds to human N3pGlu amyloid β-peptide and its use in treating diseases involving β-amyloid. Background Technology

[0004] Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and the most prevalent type of dementia in old age. It is mainly characterized by abnormal brain function and personality / behavioral changes, such as cerebral cortex atrophy, hippocampal atrophy, memory loss, difficulty expressing ideas, confusion, and impaired judgment, severely impacting patients' daily lives (FRATIGLIONI L, QIU C et al., Experimental Gerontology, 2009 Jan-Feb; 44(1-2):46-50). The risk of developing the disease increases with age. The etiology of AD is complex, and the exact pathogenesis remains unclear, but it is generally considered to be the result of a combination of factors including aging, genetics, and environment. According to data published in 2020, an estimated 15.07 million people aged 60 and above in China suffer from dementia (prevalence 6.0%), of which approximately 9.83 million are Alzheimer's disease patients (prevalence 3.9%), demonstrating the high prevalence of AD (LV B, LIANG L et al., International Journal of Public Health, 2023 Feb 3:68:1605129). Furthermore, according to "The China Alzheimer Report 2022," the age-standardized mortality rate for AD and other dementia patients in my country in 2019 was 23 (cases / 100,000 people), making it the fifth leading cause of death in my country, further highlighting the high mortality rate of AD. Given the high prevalence and mortality of AD, the current number of marketed treatments is limited and their effectiveness is lacking; therefore, there remains a significant unmet need to develop treatments that can stop or delay disease progression.

[0005] Aβ (β-amyloid protein) deposition in the brain is one of the pathological manifestations of Alzheimer's disease. Under normal physiological conditions, the production and clearance of Aβ are in a dynamic balance. However, under pathological conditions, the production of Aβ increases or the clearance decreases, which disrupts the balance. Aβ is excessively deposited in the brain, forming plaques, which leads to a series of pathological processes, such as oxidative stress, neurofibrillary tangles, and mitochondrial dysfunction (Oh, ES, Troncoso, JC & Fangmark Tucker, SM Neuromol Med, 2008; 10(3):195-207).

[0006] Deposits in brain plaques consist of a heterogeneous mixture of various Aβ peptides. N3pGlu Aβ (also known as N3pE, pE3-X, Aβp3-X, or AβN3pE-42) is an N-terminal truncated form of the Aβ peptide found in brain plaques. N3pGlu Aβ lacks the first two N-terminal amino acid residues of human Aβ, and the glutamate derived from the third amino acid position is pyroglutamicized (i.e., pyroglutamic acid, Pyr). Although N3pGlu Aβ peptide is a minor component of Aβ deposited in the brain, studies have shown that it has a strong tendency to aggregate and accumulates early in the depositional cascade process. Current testing has revealed that N3pGlu Aβ exists only in plaques and not in other forms, demonstrating high specificity and making it a promising target for treating diseases caused by Aβ deposition (BAYER TA et al., Molecular psychiatry, 2022 Apr; 27(4):1880-1885).

[0007] Antibodies targeting N3pGlu Aβ already exist in this field, demonstrating superior plaque clearance capabilities compared to other clinically available antibodies targeting Aβ, such as the marketed Donanemab monoclonal antibody. Another N3pGlu Aβ-targeting monoclonal antibody, Remternetug (patent number: CN110582511B, name 201c), is currently in Phase III clinical trials. According to its Phase I clinical data, its plaque clearance efficacy is superior to that of the marketed Lecanemab and Donanemab. However, developing molecules superior to Remternetug to further improve the properties of this antibody molecule, including higher affinity, better Aβ clearance efficacy, superior pharmacokinetic properties, and better stability under physiological conditions, to fill the significant gap in Alzheimer's disease treatment remains urgently needed. Summary of the Invention

[0008] This disclosure provides an antibody (or anti-N3pGlu Aβ antibody) that binds to N3pGlu Aβ, which can specifically bind to human N3pGlu Aβ and has higher antigen-antibody affinity, better in vitro Aβ polymer clearance effect and better stability under physiological conditions compared with Remternetug.

[0009] One aspect of this disclosure is to provide an antibody that binds to N3pGlu Aβ, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR region sequence of a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO:11, 12 or 13, and / or, the light chain variable region comprises an LCDR region sequence of a light chain variable region (VL) with an amino acid sequence as shown in SEQ ID NO:14. In one specific embodiment, the antibody that binds to N3pGlu Aβ of this disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR1 sequence as shown in SEQ ID NO:3, an HCDR2 sequence as shown in SEQ ID NO:4, and an HCDR3 sequence as shown in SEQ ID NO:5, 6 or 7, and / or, the light chain variable region comprises an LCDR1 sequence as shown in SEQ ID NO:8, an LCDR2 sequence as shown in SEQ ID NO:9, and an LCDR3 sequence as shown in SEQ ID NO:10.

[0010] One aspect of this disclosure is to provide an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ, wherein the antibody or antigen-binding fragment thereof is specifically capable of binding to human N3pGlu Aβ, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are SEQ ID NO: 3, 4, 7, 8, 9, and 10, respectively.

[0011] In one specific embodiment, the antibody binding to N3pGlu Aβ comprises a heavy chain variable region as shown in SEQ ID NO:11, 12 or 13, and / or a light chain variable region as shown in SEQ ID NO:14.

[0012] One aspect of this disclosure is to provide an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are respectively the following amino acid sequences: SEQ ID NO:13 and 14.

[0013] In one specific embodiment, the antibody binding to N3pGlu Aβ has the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:11 and the light chain variable region (VL) amino acid sequence of SEQ ID NO:14. In another specific embodiment, the antibody binding to N3pGlu Aβ has the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:12 and the light chain variable region (VL) amino acid sequence of SEQ ID NO:14. In yet another specific embodiment, the antibody binding to N3pGlu Aβ has the heavy chain variable region (VH) amino acid sequence of SEQ ID NO:13 and the light chain variable region (VL) amino acid sequence of SEQ ID NO:14.

[0014] One aspect of this disclosure is to provide an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR region sequence of the heavy chain variable region (VH) as shown in SEQ ID NO:13, and the light chain variable region comprises an LCDR sequence of the light chain variable region (VL) as shown in SEQ ID NO:14.

[0015] The antibody that binds N3pGlu Aβ disclosed herein may be a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a murine antibody, a humanized antibody, a chimeric antibody, a modified antibody, a fully human antibody, a full-length antibody, a heavy chain antibody, a nanobody, Fab, Fv, scFv, F(ab')2, a linear antibody, or a heavy chain single-domain antibody.

[0016] The antibody that binds to N3pGlu Aβ disclosed herein may be in the form of IgG1, IgG2, IgG3 or IgG4.

[0017] In one specific embodiment, the antibody that binds to N3pGlu Aβ disclosed herein may further include a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains an Fc; more preferably, the Fc is derived from a mouse or a human; even more preferably, the sequence of the Fc is a natural or modified variant.

[0018] In some embodiments, the antibody binding N3pGlu Aβ comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of the heavy chain is SEQ ID NO:15, 16, or 17, and the amino acid sequence of the light chain is SEQ ID NO:18. In a specific embodiment, an antibody binding N3pGlu Aβ is provided, wherein the amino acid sequence of the heavy chain is SEQ ID NO:15, and the amino acid sequence of the light chain is SEQ ID NO:18. In another specific embodiment, an antibody binding N3pGlu Aβ is provided, wherein the amino acid sequence of the heavy chain is SEQ ID NO:16, and the amino acid sequence of the light chain is SEQ ID NO:18. In yet another specific embodiment, an antibody binding N3pGlu Aβ is provided, wherein the amino acid sequence of the heavy chain is SEQ ID NO:17, and the amino acid sequence of the light chain is SEQ ID NO:18.

[0019] In this disclosure, the antigen-binding fragment of the antibody that binds to N3pGlu Aβ is as described above.

[0020] This disclosure provides a fusion protein, wherein one fused portion comprises the antibody that binds N3pGlu Aβ.

[0021] This disclosure also provides a fusion protein comprising the above-described antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ.

[0022] This disclosure also provides a bispecific or multispecific antibody, wherein one antigen-binding domain comprises the antibody that binds to N3pGlu Aβ.

[0023] This disclosure also provides a bispecific or multispecific antibody, wherein one antigen-binding domain comprises an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ as described in any one of claims 1-4.

[0024] In one embodiment, this disclosure provides a nucleic acid molecule that encodes the antibody that binds to N3pGlu Aβ.

[0025] In one embodiment, this disclosure provides a nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ.

[0026] This disclosure provides a carrier containing the aforementioned nucleic acid molecule.

[0027] This disclosure also provides a host cell containing the aforementioned nucleic acid molecule or vector; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, an NSO cell, an Expi293 cell, or a HEK293 cell.

[0028] This article provides a method for preparing antibodies, fusion proteins, bispecific antibodies, or multispecific antibodies that bind to N3pGlu Aβ as described above. The method includes culturing the host cells in a culture medium; preferably, the method further includes a step of purifying and recovering the antibody.

[0029] This article provides a method for preparing antibodies or antigen-binding fragments, fusion proteins, bispecific antibodies, or multispecific antibodies that bind to N3pGlu Aβ as described above. The method includes culturing the host cells in a culture medium; preferably, the method further includes a step of purifying and recovering the antibody.

[0030] This disclosure provides the use of the aforementioned N3pGlu Aβ-binding antibodies, fusion proteins, bispecific antibodies, multispecific antibodies, nucleic acid molecules, vectors, and / or host cells for the preparation of medicaments for treating, alleviating, and / or preventing diseases. This disclosure also provides the use of the aforementioned N3pGlu Aβ-binding antibodies or their antigen-binding fragments, fusion proteins, bispecific antibodies, multispecific antibodies, nucleic acid molecules, vectors, and / or host cells for the preparation of medicaments for treating, alleviating, and / or preventing diseases. Preferably, the disease is a disease caused by β-amyloid protein; more preferably, the disease is selected from Alzheimer's disease (AD), Down syndrome, or cerebral amyloid angiopathy (CAA).

[0031] This disclosure also provides pharmaceutical compositions comprising the aforementioned antibodies that bind N3pGlu Aβ, fusion proteins, bispecific antibodies, multispecific antibodies, nucleic acid molecules, vectors, and / or host cells.

[0032] This disclosure also provides pharmaceutical compositions comprising the aforementioned antibody binding N3pGlu Aβ or an antigen fragment thereof, a fusion protein, a bispecific antibody, a multispecific antibody, a nucleic acid molecule, a vector, and / or a host cell.

[0033] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0034] In one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0035] In one embodiment, the pharmaceutical composition is used to treat, alleviate, and / or prevent a disease. Preferably, the disease is a disease caused by β-amyloid protein; more preferably, the disease is selected from Alzheimer's disease (AD), Down syndrome, or cerebral amyloid angiopathy (CAA).

[0036] This disclosure also provides the use of the antibodies, nucleic acid molecules, vectors, and / or host cells that bind to N3pGlu Aβ for the breakdown of β-amyloid plaques.

[0037] The antibody that binds to N3pGlu Aβ provided in this disclosure has significantly superior antigen affinity, specificity, and Aβ clearance ability, while also exhibiting good stability. Attached Figure Description

[0038] Figure 1 shows the experimental results of the binding affinity of different antibodies to soluble N3pGlu Aβ;

[0039] Figure 2 shows the experimental results of the binding affinity of different antibodies to aggregated N3pGlu Aβ;

[0040] Figure 3A shows the phagocytosis experiment of BV2 microglia, demonstrating the experimental results of the antibody against N3pGlu Aβ promoting the phagocytosis of AβN3pE-42 polymers by BV2 cells;

[0041] Figure 3B shows the phagocytosis experiment of BV2 microglia, demonstrating the experimental results of the antibody against N3pGlu Aβ promoting the phagocytosis of BV2 cells on the mixture of AβN3pE-42 and Aβ1-42 aggregates.

[0042] Figure 4A shows the nonspecific binding levels of the anti-N3pGlu Aβ antibody to fibrinogen, double-stranded DNA, and low-density lipoprotein;

[0043] Figure 4B shows the nonspecific binding levels of the anti-N3pGlu Aβ antibody to fibronectin, human serum albumin, and lipopolysaccharide;

[0044] Figure 5 shows the non-specific binding levels of the anti-N3pGlu Aβ antibody to CHO-S cells and HEK293F cells;

[0045] Figure 6 shows the half-life of the anti-N3pGlu Aβ antibody in mice transgenic with the FcRn gene. Detailed Implementation

[0046] the term

[0047] To facilitate understanding of the contents of this disclosure, the following technical and scientific terms are defined.

[0048] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0049] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each publication, patent or patent application has been specifically and individually indicated to be incorporated herein by reference.

[0050] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0051] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0052] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0053] As used in this article, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH), or heavy chain constant region (CH).

[0054] Five main classes of antibodies are known in this art: IgA, IgD, IgE, IgG, and IgM, with their corresponding heavy chain constant domains designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chain of antibodies from any vertebrate species can be identified as one of two distinctly different types based on the amino acid sequence of their constant domains, designated κ and λ.

[0055] In the case of IgG, IgA, and IgD antibodies, this heavy chain constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0056] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antigen-binding site, giving the complete IgG antibody two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4. The CDR amino acid residues of the antibodies or antigen-binding fragments described in this disclosure conform to the known Kabat numbering rules in number and position (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any nomenclature system based on the antibody sequences of this disclosure are clearly kept within the scope of this disclosure.

[0057] The term "Fc" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0058] The term "antibody" in this disclosure can include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding portion) or its single chain, and can also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.). The term "antigen-binding fragment" refers to a polypeptide fragment in an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its antigenic epitope, or a fusion protein product further derived from this fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0059] In this article, "antibody" can be used in the broadest sense, including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0060] In one embodiment, the antibody disclosed herein comprises κLC and IgG HC. In a specific embodiment, the antibody disclosed herein is IgG1.

[0061] The term "Fab fragment" includes the variable regions of the heavy chain and the light chain, and also includes the constant region of the light chain and the first constant region CH1 of the heavy chain; it is a monovalent antibody fragment. The term "F(ab')2 fragment" contains two Fab fragments and a hinge region; it is a bivalent antibody fragment.

[0062] The term "Fd fragment" generally includes the heavy chain variable region and the constant region CH1; the term "Fv fragment" contains the antibody heavy chain variable region and the light chain variable region, but no constant region, and is the smallest antibody fragment with all antigen binding sites.

[0063] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0064] The term "fusion protein" refers to a larger molecule formed by linking different polypeptides / proteins together through genetic recombination or chemical methods. Linkers can be used for this linking, or not.

[0065] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0066] The term "chimeric antibody" is a construct in which a portion of the heavy and / or light chains is identical or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of this or these chains is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, or in fragments of such antibodies. In a narrower sense, a chimeric antibody comprises all or most of selected murine heavy and light chain variable regions operatively linked to human light and heavy chain constant regions. Constant region sequences, or variants or derivatives thereof, can be operatively associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide a full-length antibody that can be used on its own or incorporated into this disclosure.

[0067] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues of the receptor's core sequence (CDR) are replaced by residues of a CDR from a non-human species (donor antibody) possessing the desired specificity, affinity, and performance, such as mice, rats, rabbits, or primates. In some cases, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain circumstances, "reversion mutations" can be introduced into humanized antibodies where residues in one or more frame regions (FRs) of the variable region of the recipient human antibody are replaced by corresponding residues from a non-human species donor antibody. Such reversion mutations can help maintain the appropriate three-dimensional conformation of one or more grafted CDRs and thus improve affinity and antibody stability. Antibodies from a variety of donor species can be used, including but not limited to mice, rats, rabbits, or non-human primates. Additionally, humanized antibodies may contain novel residues not found in the recipient antibody or the donor antibody to further improve antibody performance.

[0068] The "KD" or "Kd" mentioned in this disclosure refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. In a specific embodiment, surface plasmon resonance (SPR) technology is used... Measured in an 8K (Cytiva) instrument.

[0069] The term "nucleic acid molecule" as used in this disclosure refers to a DNA molecule or an RNA molecule. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0070] The term "vector" as used in this disclosure refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In specific embodiments, the vector is also referred to as a "plasmid," which is a circular double-stranded DNA loop into which another DNA segment can be linked.

[0071] The terms "nucleic acid molecule encoding," "encoding DNA sequence," and "encoding DNA" refer to the sequence of deoxyribonucleotides along a deoxyribonucleic acid (DNA) chain. This sequence of deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0072] The term "N3pGlu Aβ" refers to the amino acid sequence from Pyr to Aβ 42 after pyroglutamic acid cyclization of Glu at position 3 in the amino acid sequence Aβ1-42 (SEQ ID NO:1). "N3pGlu Aβ" and "AβN3pE-42" refer to the same sequence (SEQ ID NO:2) and can be used interchangeably herein.

[0073] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0074] The “diseases or conditions caused by β-amyloid protein” as described in this disclosure refer to diseases or conditions caused by or related to β-amyloid protein, including but not limited to diseases and conditions caused by the presence or activity of β-amyloid protein in the form of Aβ monomers, protofibrils, or polymers, or any combination thereof, such as neurodegenerative diseases (e.g., Alzheimer's disease, mild cognitive impairment, frontotemporal dementia, Lewy body disease, Parkinson's disease, Pick's disease, Down syndrome, cerebral amyloid angiopathy (CAA), Bewak's disease, etc.), and various eye diseases caused by β-amyloid protein deposition (e.g., macular degeneration, drusen-associated optic neuropathy, glaucoma, cataracts, etc.).

[0075] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained herein are present in a biologically effective form and do not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. When a "pharmaceutical composition" exists as a combination of individual formulations containing two or more different active ingredients, it can be administered simultaneously, sequentially, separately, or at intervals, with the aim of exerting the biological activity of multiple active ingredients together for the treatment of a disease.

[0076] The "amyloid beta peptide disease" in this disclosure refers to diseases pathologically characterized by Aβ deposition in the brain or cerebral blood vessels. This includes diseases such as Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy.

[0077] Example

[0078] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments, unless specific conditions are specified, are generally performed under conventional conditions known in the art or as recommended by the manufacturer.

[0079] Example 1: Engineering of anti-N3pGlu Aβ antibody

[0080] 1. Phage library construction

[0081] The nucleic acid sequence encoding the light chain variable region sequence VL (SEQ ID NO:20) of the Remternetug antibody (amino acid sequence information from CN110582511B) was assembled into the pCANTAB-5E-Fab plasmid using molecular cloning methods to obtain the RemVL-pCANTAB-5E-Fab plasmid with the light chain VL encoding nucleic acid sequence. All oligonucleotide primers required for amplifying the VH (SEQ ID NO:19) coding nucleic acid sequence of Remternetug were synthesized. For primers involving the HCDR3 sequence EGSGSYYNG (SEQ ID NO:21), NNK (N=A / C / G / T, K=G / T) saturation mutations were introduced at three deoxyribonucleotide positions. The VH gene was synthesized using 120 primers with mutations via Assembly PCR. Each sequence contained three NNK mutation sites. The VH coding nucleic acid sequences with saturation mutations were then ligated into the RemVL-pCANTAB-5E-Fab vector using restriction endonucleases and T4 DNA ligase. The desalted ligation product was electroporated into SS320 *E. coli* (manufacturer: Lucigen, catalog number 60512-1), resulting in a saturation mutant library with a capacity of 4.17E7. Fifty randomly selected single clones were sequenced to check the assembly accuracy.

[0082] 2. Phage screening

[0083] The constructed saturated mutant library was cultured, followed by phage infection, PEG / NaCl precipitation, and PBS resuspending to obtain a packaged phage library. This library underwent two rounds of solid panning using streptavidin-based magnetic beads. Before each round of screening, a final concentration of 100 μM Aβ1-42 was added for blocking to prevent antibody binding to Aβ1-42. The first and second rounds of screening involved the addition of 50 nM and 5 nM biotin-labeled AβpE3-16 (synthesized by Shanghai Jier Biochemical, amino acid sequence Pyr-FRHDSGYEVHHQK-Biotin, SEQ ID NO:22), respectively. After two rounds of screening, positive clones were selected using phage ELISA and then subjected to Sanger sequencing to obtain the antibody sequences.

[0084] Example 2: Expression and purification of engineered anti-N3pGlu Aβ antibody

[0085] 1. Molecular construction

[0086] For molecules that are positive for phage ELISA, primers were designed to construct the nucleic acid fragments encoded by the VH and VL of each antibody via PCR. These fragments were then homologously recombinated with the expression vector pcDNA3.4 (containing a signal peptide and a constant region gene (CH1-Fc / CL) fragment) to construct expression plasmids containing nucleic acids encoded by the antibody heavy and light chains. After sequencing verification, plasmids were prepared.

[0087] 2. Expression and purification of humanized antibodies

[0088] Use ExpiCHO TM Antibody expression was performed using an expression system (Thermo Scientific, catalog number A29133). Plasmids encoding the antibody heavy and light chains were transfected into ExpiCHO-S cells at a ratio of 1:1.5. Eight days after transfection, the expression supernatant was collected, and impurities were removed by high-speed centrifugation. Antibody purification was performed using Protein A magnetic beads (Nanjing GenScript Technology Co., Ltd., catalog number L00695). The target antibody was eluted with 20 mM acetic acid and neutralized with 1 M Tris-HCl (pH 8.0) to obtain the target antibody. After appropriate concentration, the eluted sample was transferred to PBS buffer and aliquoted for use. The final purified antibody was analyzed for purity by SDS-PAGE, SEC-HPLC, and A280 concentration determination.

[0089] Example 3: Binding affinity for soluble N3pGlu Aβ

[0090] A. ELISA method to detect the binding of the test antibody to soluble N3pGlu Aβ.

[0091] The affinity of the antibody for soluble N3pGlu Aβ was detected by ELISA to screen for high-affinity anti-N3pGlu Aβ antibodies. 1 mg of N3pGlu Aβ peptide was dissolved in 400 μL HFIP (manufacturer: Antech Chemicals, catalog number 920-66-1) and allowed to evaporate overnight at room temperature. After evaporation, 1 mL of DMSO was added for complete dissolution, and the solution was aliquoted for use. The soluble N3pGlu Aβ dissolved in DMSO was then added to 20 times its volume of PBS buffer and sonicated until fully dissolved. Subsequently, a standard ELISA assay was performed, with 0.2 μg of the treated N3pGlu Aβ per well (ELISA plate manufacturer: Thermoscientific, catalog number 446469) and incubated at room temperature for 2 hours. After incubation, the plate was washed four times with PBST (PBS buffer containing 0.5% Tween 20) and blocked with blocking buffer (PBS buffer containing 3% BSA) at room temperature for 2 hours. After blocking, the sample was washed four times with PBST, and then different concentrations of the test antibody were added and incubated at room temperature for 1 hour. After incubation, the sample was washed four times with PBST, and then incubated for 1 hour with HRP-labeled anti-human Fc secondary antibody (manufacturer: Nanjing GenScript Technology Co., Ltd., catalog number: A01854). Subsequently, the sample was washed five times with PBST, and then 100 μL of TMB chromogenic solution (manufacturer: Cell Signaling Technology, catalog number: 7004P6) was added for 15 minutes of light-protected color development. Finally, 100 μL of 1M H2SO4 was added to terminate the reaction, and the absorbance at 450 nm was measured using a microplate reader.

[0092] The experimental results are shown in Table 1 and Figure 1 below. The selected anti-N3pGlu Aβ antibodies are RemH1 (HCDR1-3 sequences are SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; LCDR1-3 sequences are SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; heavy chain variable region sequence is SEQ ID NO:11; light chain variable region sequence is SEQ ID NO:14; heavy chain sequence is SEQ ID NO:15; light chain sequence is SEQ ID NO:18), RemH3 (HCDR1-3 sequences are SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:6; LCDR1-3 sequences are SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; heavy chain variable region sequence is SEQ ID NO:12; light chain variable region sequence is SEQ ID NO:14; heavy chain sequence is SEQ ID NO:16; light chain sequence is SEQ ID NO:18) and RemH16 (HCDR1-3 sequences are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14; heavy chain sequence is SEQ ID NO:16; light chain sequence is SEQ ID NO:18) and RemH16 (HCDR1-3 sequences are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14; heavy chain sequence is SEQ ID NO:16; light chain sequence is SEQ ID NO:18) are also shown in Table 1 and Figure 1. The sequences NO:3, SEQ ID NO:4, and SEQ ID NO:7, and the LCDR1-3 sequences are SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; the heavy chain variable region sequence is SEQ ID NO:13, the light chain variable region sequence is SEQ ID NO:14; the heavy chain sequence is SEQ ID NO:17, and the light chain sequence is SEQ ID NO:18) have significantly higher binding affinity to soluble N3pGlu Aβ peptides than Remternetug antibody. The binding affinity of RemH1, RemH3, and RemH16 to soluble N3pGlu Aβ peptides is 22.5 times, 23.2 times, and 29.9 times that of Remternetug antibody, respectively.

[0093] Table 1. Binding affinity of antibodies to soluble N3pGlu Aβ

[0094] B. Surface plasmon resonance technique for detecting the binding and dissociation kinetics of the antibody.

[0095] use The 8K (manufactured by Cytiva) instrument uses surface plasmon resonance to measure the binding and dissociation kinetics and affinity of the test antibody with the soluble N3pGlu Aβ peptide. Firstly, in... Approximately 8000 RU of the target antibody was immobilized on the CM5 chip via amino-coupled polymerization. Binding kinetics and affinity were measured by flowing through soluble N3pGlu Aβ peptide serially diluted 2-fold from 500 nM down to 15.6 nM. The experimental temperature was 25 °C, and the buffer used was HBS-EP+ buffer (Cytiva BR100669; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). For each cycle, the soluble N3pGlu Aβ peptide was flowed at 30 μL / min through the reference and experimental channels, bound for 3 min, and then dissociated for 10 min. The chip surface was regenerated by injecting 50 mM hydrochloric acid at a flow rate of 30 μL / min for 30 s, and this regeneration was repeated twice. After double subtraction (i.e., in each cycle, the experimental channel signal is subtracted from the reference channel signal, and the sample signal is then subtracted from the blank signal), the data was kinetically fitted using Biacore Insight Evaluation software. A 1:1 binding model was used for the fitting model to obtain k. on k off And calculate the KD value.

[0096] The experimental results are shown in Table 2 below. The antibodies RemH1, RemH3, and RemH16 obtained by screening for anti-N3pGlu Aβ showed significantly higher binding affinity to soluble N3pGlu Aβ peptides than Remternetug antibody. The binding affinity of RemH1, RemH3, and RemH16 to soluble N3pGlu Aβ peptides was 2.94 times, 37.5 times, and 5.07 times that of Remternetug antibody, respectively.

[0097] Table 2. Binding kinetics and affinity of antibodies to soluble N3pGlu Aβ

[0098] Example 4: Binding affinity for aggregated N3pGlu Aβ

[0099] First, aggregated N3pGlu Aβ peptides were prepared. N3pGlu Aβ peptide powder was dissolved in 10 mM NaOH solution (pH > 10, containing 0.05% Tween 20) to a concentration of 0.43 mg / mL. After vortexing for 2 min, an equal volume of 2×PBS was added, and the mixture was incubated overnight at 37°C. The supernatant was then removed by centrifugation, and the precipitate was the aggregated N3pGlu Aβ peptide.

[0100] Next, the binding affinity of the antibody to aggregated N3pGlu Aβ was detected using an ELISA method. The specific detection method is the same as in Part A of Example 3.

[0101] The experimental results are shown in Table 3 and Figure 2. The binding affinity of the anti-N3pGlu Aβ antibodies RemH1, RemH3, and RemH16 to the aggregated N3pGlu Aβ peptide was significantly improved compared to the Remternetug antibody. Specifically, the binding affinity of RemH1, RemH3, and RemH16 to the aggregated N3pGlu Aβ peptide was 8.00 times, 5.66 times, and 6.49 times that of the Remternetug antibody, respectively.

[0102] Table 3. Binding affinity of antibodies to aggregated N3pGlu Aβ

[0103] Example 5: Anti-N3pGlu Aβ antibody promotes phagocytosis of Aβ polymers by BV2 cells.

[0104] BV2 cells in logarithmic growth phase (manufactured by Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60922) were used at a density of 1.0 × 10⁶ cells per well. 5 Cells were evenly seeded into 24-well plates and incubated overnight at 37°C in a 5% CO2 cell culture incubator. After 24 hours, the antibody against N3pGlu Aβ and the negative control (using target-independent RSV antibody as a negative control) were diluted with DMEM medium and mixed thoroughly with 10 μM Alexa Flour 488-labeled antigen (100% AβN3pE-42 aggregates or 15% AβN3pE-42 + 85% Aβ1-42 aggregates). The mixture was then incubated at 37°C for 1 hour. The supernatant was then discarded, and the cells were gently washed three times with pre-chilled PBS buffer. Pre-chilled trypsin was then added for digestion for 20 minutes, and digestion was terminated with DMEM medium containing 10% FBS. The cells were then washed three times with pre-chilled PBS, and the cells were collected for flow cytometry analysis.

[0105] The experimental results are shown in Figures 3A and 3B. Antibodies RemH1, RemH3, and RemH16 against N3pGlu Aβ effectively promoted the phagocytosis of antigens by BV2 cells, and their phagocytic capacity was significantly stronger than that of Remternetug. Furthermore, the percentage of antibody-mediated phagocytosis increased with increasing antibody concentration, exhibiting a dose-dependent relationship.

[0106] Example 6: Thermal stability test

[0107] A. Differential Scanning Calorimetry (DSC) Analysis

[0108] Differential scanning calorimetry (DSC) is a widely recognized analytical technique for characterizing the thermal stability of the higher-order structure of biological samples. It can measure and analyze the heat capacity changes of samples in real time during controlled heating, obtain thermodynamic characterization parameters of sample thermal stability, and assess the higher-order structure and thermal stability of protein drugs.

[0109] The thermal stability of the anti-N3pGlu Aβ antibody disclosed herein was determined using differential scanning calorimetry. The scanning temperature ranged from 20℃ to 100℃, with a scanning rate of 90℃ / hr. The test sample was diluted with blank buffer (PBS buffer) to a concentration of 1.0 mg / mL, and the blank buffer was used as a blank control. The Tg of the test antibody was analyzed and calculated using software. m value.

[0110] The experimental results are shown in Table 4 below. Compared with Remternetug, the Tm values ​​of RemH3 and RemH16 did not change significantly, while the Tm value of RemH1... m The value of 1 increased significantly.

[0111] Table 4. Tm values ​​of the antibodies to be tested

[0112] B. Thermal stability under stress conditions

[0113] To determine the stability of the modified molecule under stress conditions (i.e., elevated temperature), the following experiment was designed for verification. The antibody to be tested was transferred to PBS buffer to adjust the antibody concentration to 10 mg / mL. The sample was sealed and incubated at 65°C for 2 hours, followed immediately by cooling on ice. Protein aggregates were removed by centrifugation at 1000 rpm for 5 minutes, and the supernatant was transferred to a new EP tube for concentration determination. Using the unstressed sample as a reference, antigen-antibody binding activity was detected using ELISA, and the percentage of remaining activity after stress was calculated.

[0114] The experimental results are shown in Table 5 below. The binding activity percentages of the anti-N3pGlu Aβ antibodies RemH1, RemH3, RemH16, and Remternetug under stress conditions did not change significantly compared with those before stress, indicating that the stability of RemH1, RemH3, and RemH16 molecules under stress conditions is similar to that of Remternetug, and all of them have good stability.

[0115] Table 5: Percentage of antigen-antibody binding activity under stress conditions

[0116] Example 7: Stability test under physiological conditions

[0117] To further simulate the performance of the antibody under normal physiological conditions, the antibody was transferred to PBS buffer (pH=7.4) and placed at 37°C for 2 weeks (14 days) and 4 weeks (28 days). The antibody charge distribution after 2 weeks and 4 weeks was detected using iCIEF to determine whether the antibody was stable under normal physiological conditions.

[0118] The experimental results are shown in Table 6 below. After being placed at 37℃ for 2 and 4 weeks, the proportion of the acidic peak of the antibodies RemH1 and RemH16 against N3pGlu Aβ was significantly smaller than that of Remternetug, while the proportion of the main peak was significantly higher. This further demonstrates that the RemH1 and RemH16 antibodies invented in this study are significantly more stable than the Remternetug antibody under physiological conditions.

[0119] Table 6. Charge distribution of antibodies after different storage times under physiological conditions

[0120] Example 8: Nonspecific binding

[0121] A. Non-specific binding to proteins, DNA, and polysaccharides.

[0122] To determine the specificity of the anti-N3pGlu Aβ antibody and verify its non-specific binding to the protein, an ELISA method was used. First, 20 μg of the test substance was coated onto a plate overnight at 4°C. The coating substance could be fibrinogen (Sigma, catalog number F3879), human serum albumin (Sigma, catalog number A9511), fibronectin (Sigma, catalog number F2006), low-density lipoprotein (Yeasen, catalog number 20613ES05), lipopolysaccharide (Thermo Scientific, catalog number 00-4976-93), or double-stranded DNA (Sigma, catalog number D4522). The plate was then washed four times with PBST. Next, the plate was blocked with blocking buffer at room temperature for 1 hour, followed by four washes with PBST. After washing, 100 nM of the antibody solution was added to each well, and the plate was incubated at room temperature for 2 hours, followed by four washes with PBST. Then, secondary antibody against human Fc-HRP was added, and the mixture was incubated at room temperature for 1 hour. Subsequently, the mixture was washed 5 times with PBST, and 100 μL of TMB chromogenic solution (manufacturer: Cell Signaling Technology, catalog number 7004P6) was added for incubation in the dark for 15 minutes. Then, 100 μL of 1MH2SO4 was added to terminate the reaction, and the absorbance at 450 nm was measured using a microplate reader.

[0123] The experimental results are shown in Figures 4A and 4B. The anti-N3pGlu Aβ antibody obtained by screening in this publication showed no increase in nonspecificity of RemH3 for fibrinogen, except for an increase in the nonspecificity of other molecules, including RemH1 and RemH16. The specificity effect was no less than that of Remternetug.

[0124] B. Non-specific binding to cells

[0125] To detect the non-specific binding of the anti-N3pGlu Aβ antibody disclosed herein to cells, suspension cells CHO-S cells and HEK293F cells were used as target cells. First, the concentrations of CHO-S cells and HEK293F cells were adjusted to 4 × 10⁻⁶. 6 Add the antibody to be tested (final concentration 100 nM) and 2 × 10⁻⁶ mL to a 96-well plate. 5 Target cells, with a total volume of 100 μL, were incubated at 4°C for half an hour. After incubation, the cells were washed three times with pre-chilled FACS buffer (PBS buffer containing 2% FBS) at 4°C, and then anti-Human Fc secondary antibody with PE fluorescence (manufacturer: Biolegend, catalog number 410708) was added, followed by incubation at 4°C for half an hour. After incubation, the cells were washed three times again with pre-chilled FACS buffer at 4°C, and finally resuspended in FACS buffer to adjust the cell concentration. Cell fluorescence intensity was detected by flow cytometry, and the data were processed and analyzed.

[0126] The experimental results are shown in Figure 5. In the non-specific binding assay with CHO-S cells, the anti-N3pGlu Aβ antibodies RemH1, RemH3, and RemH16 obtained in this study did not show an increase in non-specificity compared to Remternetug. In the non-specific binding assay with HEK293F cells, the non-specificity of RemH16 was similar to that of Remternetug, while the non-specificity of RemH1 and RemH3 showed a slight increase.

[0127] Example 9: Half-life detection of anti-N3pGlu Aβ antibody

[0128] Eight 6-8 week old B-FcRn mice (Biocytok Jiangsu Gene Biotechnology Co., Ltd., catalog number 110001) (half male and half female) were selected for antibody half-life detection. The mice were randomly divided into two groups, with two females and two males in each group. The drug dosage was 30 mg / kg, administered via a single tail vein injection at a volume of 10 mL / kg. Blood samples were collected from the mice at 5 min, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h, 240 h, 336 h, 504 h, 672 h, 840 h, and 1008 h post-administration. Each collection yielded 7-8 drops of fresh blood, which were allowed to stand at room temperature for 30 min, followed by centrifugation at 4500 rpm for 15 min. The supernatant was collected as serum.

[0129] The antibody concentration in serum was then detected using ELISA. The values ​​from the blank wells were subtracted from the test samples, and a drug-time curve was plotted, with serum drug concentration on the ordinate and blood collection time on the abscissa. If the serum sample was diluted before testing, the final calculation must be multiplied by the sample dilution factor to obtain the actual concentration of the sample.

[0130] The experimental results are shown in Table 7 and Figure 6. Compared with Remternetug, the half-life of the anti-N3pGlu Aβ antibody RemH16 is 1.42 times that of Remternetug, and its clearance rate (CL value) is also lower. These experimental results indicate that the pharmacokinetic properties of RemH16 are superior to those of Remternetug.

[0131] Table 7. Pharmacokinetic parameters of the antibody to be tested

[0132] Some of the sequences used in this disclosure are shown in Table 8.

[0133] Table 8. Sequence List

[0134] While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ, wherein, The antibody or its antigen-binding fragment specifically binds to human N3pGlu Aβ, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as follows: SEQ ID NO:3, 4, 7, 8, 9 and 10.

2. An antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are respectively the following amino acid sequences: SEQ ID NO:13 and 14.

3. An antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR region sequence of the heavy chain variable region (VH) as shown in SEQ ID NO:13, and the light chain variable region comprises an LCDR sequence of the light chain variable region (VL) as shown in SEQ ID NO:

14.

4. The antibody or antigen-binding fragment of N3pGlu Aβ as described in any one of claims 1-3, further comprising one or more of the following characteristics: (1) It also includes the heavy chain constant region and / or the light chain constant region; (2) It is a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; (3) It is a monoclonal antibody; (4) It is a full-length antibody, or it is a Fab, Fv, scFv, F(ab')2, linear antibody, or single-domain antibody; (5) It is in the form of IgG1, IgG2, IgG3 or IgG4.

5. A fusion protein, wherein, The fusion protein comprises an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ as described in any of the preceding claims.

6. A bispecific or multispecific antibody, wherein one antigen-binding domain comprises an antibody or antigen-binding fragment thereof that binds N3pGlu Aβ as described in any one of claims 1-4.

7. A nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ as described in any one of claims 1-4.

8. A recombinant vector comprising the nucleic acid as described in claim 7.

9. A host cell comprising the recombinant vector as claimed in claim 8 or the nucleic acid as claimed in claim 7.

10. The host cell as described in claim 9, wherein the host cell is selected from prokaryotic cells and eukaryotic cells.

11. The host cell of claim 10, wherein the prokaryotic cell is Escherichia coli, and the eukaryotic cell is yeast or a mammalian cell.

12. The host cell of claim 11, wherein the mammalian cell is selected from CHO cells, NSO cells, Expi293 cells and HEK293 cells.

13. A method for preparing an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ as described in any one of claims 1-4, comprising: The host cell as described in any one of claims 9-12 is cultured under suitable conditions, and the expression product is purified from said cell.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to N3pGlu Aβ as described in any one of claims 1-4, or comprising a fusion protein as described in claim 5, or comprising a bispecific antibody or a multispecific antibody as described in claim 6, or comprising a nucleic acid as described in claim 7, or comprising a recombinant vector as described in claim 8, or comprising a host cell as described in any one of claims 9-12.

15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, further comprising additional therapeutic agents.

17. Use of an antibody or antigen-binding fragment thereof that binds N3pGlu Aβ as described in any one of claims 1-4, a fusion protein as described in claim 5, a bispecific antibody or multispecific antibody as described in claim 6, a nucleic acid as described in claim 7, a recombinant vector as described in claim 8, a host cell as described in any one of claims 9-12, or a pharmaceutical composition as described in any one of claims 14-16 in the preparation of a medicament for the prevention, delay, and / or treatment of a disease caused by β-amyloid protein.

18. The use as described in claim 17, wherein the disease is selected from Alzheimer's disease (AD), Down syndrome, and cerebral amyloid angiopathy (CAA).

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