CN118459583B - Anti-N3 pGlu amyloid beta antibodies and uses thereof - Google Patents

Abstract

The present disclosure relates to an anti-N3pGlu amyloid β antibody and its use, and a pharmaceutical composition comprising the anti-N3pGlu amyloid β antibody. The anti-N3pGlu amyloid β antibody has significantly excellent affinity, stability and specificity. In addition, the present disclosure also provides the use of the anti-N3pGlu amyloid β antibody for treating diseases caused by β-amyloid protein (e.g., Alzheimer's disease, Down syndrome or cerebral amyloid angiopathy, etc.).

Description

Anti-N3pGlu amyloid β antibody and its use

Technical Field

The present disclosure provides antibodies that bind to human N3pGlu amyloid β peptide and uses thereof in treating diseases caused by amyloid β.

Background Art

Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly and the main type of senile dementia. It is mainly manifested by atrophy of the cerebral cortex, atrophy of the hippocampus, memory loss, inarticulateness, confusion, decreased judgment and other abnormal brain functions and personality and behavioral changes, which seriously affect the daily life of patients (FRATIGLIONI L, QIU C et al., Experimental Gerontology, 2009 Jan-Feb;44(1-2):46-50). And the risk of disease increases with age. The cause of the disease is complex and the exact pathogenesis is still unclear. It is generally believed to be the result of multiple factors such as aging, genetics and environment. According to data published in the literature in 2020, an estimated 15.07 million people aged 60 and above in China suffer from dementia (prevalence rate 6.0%), of which approximately 9.83 million suffer from Alzheimer's disease (prevalence rate 3.9%), which shows that AD has a high prevalence (LV B, LIANG L et al., International journal of public health, 2023 Feb 3:68:1605129). At the same time, according to "The China Alzheimer Report 2022", the age-standardized mortality rate of AD and other dementia patients in my country in 2019 was 23 (cases/100,000 people), which has jumped to the fifth leading cause of death in my country, which shows that AD has a high mortality rate. In the case of high prevalence and mortality of AD, there are currently few therapeutic drugs on the market and they lack effectiveness. Therefore, there is still a great unmet need for the development of treatments that can stop or delay disease progression.

The deposition of Aβ (β-amyloid protein) in the brain is one of the pathological manifestations of Alzheimer's disease. Generally, under normal physiological conditions, the generation and clearance of Aβ are in a dynamic equilibrium process. However, under pathological conditions, the generation of Aβ increases or the clearance decreases, resulting in a break in the balance. Aβ is excessively deposited in the brain to form plaques, which causes a series of pathological processes, such as oxidative stress, neurofibrillary tangles, mitochondrial dysfunction, etc. (Oh, E.S., Troncoso, J.C. & FangmarkTucker, S.M. NeuromolMed, 2008;10(3):195-207).

Deposits in brain plaques are composed of a heterogeneous mixture of multiple Aβ peptides. N3pGlu Aβ (also known as N3pE, pE3-X, Aβp3-X, or AβN3pE-42) is an N-terminally truncated form of the Aβ peptide and is an Aβ peptide present in brain plaques. N3pGluAβ lacks the first two amino acid residues at the N-terminus of human Aβ and has pyroglutamylated glutamic acid (i.e., pyroglutamic acid, Pyr) derived from the glutamic acid at the third amino acid position. Although the N3pGlu Aβ peptide is a minor component of Aβ deposited in the brain, studies have shown that the N3pGlu Aβ peptide has a strong tendency to aggregate and accumulates early in the deposition cascade. Current tests have found that N3pGlu Aβ only exists in plaques and not in other forms. It has good specificity and is a good target for treating diseases caused by Aβ deposition (BAYER TA et al., Molecular psychiatry, 2022Apr;27(4):1880-1885).

Antibodies targeting N3pGlu Aβ are known in the art and show better plaque clearance ability than other antibodies targeting Aβ in the clinic, such as the Donanemab monoclonal antibody that is currently applying for marketing approval. Remternetug (patent number: CN110582511B, name 201c), another monoclonal antibody targeting N3pGlu Aβ under clinical development, is currently in Phase III clinical trials. According to the clinical data published in its Phase I, the plaque clearance effect is better than Lecanemab, which has been marketed, and Donanemab, which is currently applying for marketing approval. However, it is still urgently needed to develop molecules that are superior to Remternetug to further improve the properties of the antibody molecule, including higher affinity, better Aβ clearance effect, and better stability under physiological conditions, to fill the huge gap in the field of Alzheimer's disease treatment.

Summary of the invention

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

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

In a specific embodiment, the antibody that binds to N3pGlu Aβ comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:11, 12 or 13, and/or a light chain variable region with an amino acid sequence as shown in SEQ ID NO:14.

In a specific embodiment, the heavy chain variable region (VH) amino acid sequence of the antibody that binds to N3pGlu Aβ is SEQ ID NO: 11, and the light chain variable region (VL) amino acid sequence is SEQ ID NO: 14. In another specific embodiment, the heavy chain variable region (VH) amino acid sequence of the antibody that binds to N3pGlu Aβ is SEQ ID NO: 12, and the light chain variable region (VL) amino acid sequence is SEQ ID NO: 14. In another specific embodiment, the heavy chain variable region (VH) amino acid sequence of the antibody that binds to N3pGlu Aβ is SEQ ID NO: 13, and the light chain variable region (VL) amino acid sequence is SEQ ID NO: 14.

The antibodies binding to N3pGlu Aβ disclosed herein can be monoclonal antibodies, bispecific binding molecules, multispecific binding molecules, murine antibodies, humanized antibodies, chimeric antibodies, modified antibodies, fully human antibodies, full-length antibodies, heavy chain antibodies, nanobodies, Fab, Fv, scFv, F(ab')2, linear antibodies or heavy chain single domain antibodies.

The N3pGlu Aβ-binding antibodies of the present disclosure may be in the form of IgG1, IgG2, IgG3 or IgG4.

In a specific embodiment, the antibody binding to N3pGlu Aβ of the present disclosure may also include a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region comprises Fc; more preferably, Fc is derived from mouse or human; more preferably, the sequence of Fc is a natural or modified variant.

In some embodiments, the antibody that binds to 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 that binds to 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 that binds to 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 another specific embodiment, an antibody that binds to 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.

The present disclosure provides a fusion protein, wherein a fused part thereof comprises the antibody binding to N3pGlu Aβ.

The present disclosure also provides a bispecific antibody or a multispecific antibody, wherein one antigen binding domain comprises the antibody that binds to N3pGlu Aβ.

In one embodiment, the present disclosure provides a nucleic acid molecule encoding the antibody that binds to N3pGlu Aβ.

The present disclosure provides a vector comprising the nucleic acid molecule.

The present disclosure also provides a host cell, which comprises the 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, NS0 cell, Expi293 or HEK293 cell.

The present invention provides a method for preparing the above-mentioned antibody, fusion protein, bispecific antibody or multispecific antibody that binds to N3pGlu Aβ. The method comprises culturing the above-mentioned host cell in a culture medium; preferably, the method further comprises the step of purifying and recovering the antibody.

The present disclosure provides the use of the antibody, fusion protein, bispecific antibody, multispecific antibody, nucleic acid molecule, vector and/or host cell binding to N3pGlu Aβ for preparing a drug for treating, alleviating and/or preventing 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).

The present disclosure also provides a pharmaceutical composition comprising the aforementioned antibody, fusion protein, bispecific antibody, multispecific antibody, nucleic acid molecule, vector and/or host cell that binds to N3pGlu Aβ.

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

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

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

The present disclosure also provides use of the antibody, nucleic acid molecule, vector and/or host cell binding to N3pGlu Aβ for decomposing β-amyloid plaques.

The N3pGlu Aβ-binding antibody provided by the present disclosure has significantly excellent antigen affinity, specificity and Aβ-clearing ability, and also has good stability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an experimental result showing the binding affinity of different antibodies to soluble N3pGlu Aβ;

FIG2 is an experimental result showing the binding affinity of different antibodies to aggregated N3pGlu Aβ;

FIG3a is a BV2 microglial phagocytosis experiment, showing that the anti-N3pGlu Aβ antibody promotes the phagocytosis of AβN3pE-42 aggregates by BV2 cells;

FIG3 b is a BV2 microglial phagocytosis experiment, showing that the anti-N3pGlu Aβ antibody promotes the phagocytosis of BV2 cells on AβN3pE-42 and Aβ1-42 mixed aggregates;

FIG4 a shows the non-specific binding levels of anti-N3pGlu Aβ antibodies to fibrinogen, double-stranded DNA and low-density lipoprotein;

FIG4 b shows the non-specific binding levels of anti-N3pGlu Aβ antibodies to fibronectin, human serum albumin and lipopolysaccharide;

FIG. 5 shows the non-specific binding level of anti-N3pGlu Aβ antibodies to CHO-S cells and HEK293F cells.

DETAILED DESCRIPTION

the term

In order to make it easier to understand the contents of this disclosure, the following technical and scientific terms are defined.

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

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as those of ordinary skill in the art to which the present disclosure belongs.

Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in a range manner. Unless otherwise stated, it should be understood that numerical ranges or the manner of description in a range are only for the purpose of brevity and convenience, and should not be considered as a strict limitation of the scope of the present disclosure. Therefore, the description using a range manner should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within the range, as these sub-ranges and numerical points have been clearly written in this article. Regardless of the width of the numerical value, the above principles are equally applicable. When a range description is adopted, the range includes the endpoints of the range.

When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations 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.

The term "antibody" as used herein typically refers to a Y-shaped 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 have such a structure. Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH), or a heavy chain constant region (CH).

Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2. The light chain of an antibody from any vertebrate species can be identified as one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain.

In the case of IgG, IgA and IgD antibodies, the heavy chain constant region comprises 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 proline- and cysteine-rich segment of variable length. Each class of antibodies further comprises inter- and intra-chain disulfide bonds formed by paired cysteine residues.

The term "variable region" or "variable domain" shows significant variation in amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light chain/heavy chain pair forms an antigen binding site, so that a complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region of the heavy chain (VH) and the variable region of the light chain (VL) domain each contain three regions of extreme variability, known as hypervariable regions (HVRs), or more commonly, complementarity determining regions (CDRs), and each of VH and VL has four framework regions FR, represented by FR1, FR2, FR3, and FR4, respectively. Therefore, CDR and FR sequences usually 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 number and position of the CDR amino acid residues of the antibodies or antigen-binding fragments described in the present disclosure conform to the known Kabat numbering rules (Kabat, et al., Ann. NY Acad. Sci. 190: 382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)). Other naming and numbering systems, such as Chothia, IMGT or AHo, are also known to those skilled in the art. Therefore, based on the antibody sequences disclosed in the present disclosure, humanized antibodies containing one or more CDRs derived from any naming system are clearly maintained within the scope of the present disclosure.

The term "Fc" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to 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.

The "antibody" in the present disclosure may include a complete antibody (e.g., a full-length monoclonal antibody) and any antigen-binding fragment thereof (i.e., antigen-binding portion) or a single chain thereof, and may also include a product having antigen-specific binding ability formed by modification (e.g., connection to other peptide segments, functional unit rearrangement, etc.) based on a complete antibody or its antigen-binding fragment or its single chain. The term "antigen-binding fragment" refers to a polypeptide fragment in an immunoglobulin or antibody that specifically binds 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 antigen-binding fragments thereof 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 antibodies or multispecific antibodies formed by antibody fragments, etc.

The term "antibody" as used herein may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including mutant proteins and variants thereof), and the like.

In one embodiment, the antibodies of the present disclosure comprise a kappa LC and an IgG HC. In a specific embodiment, the antibodies of the present disclosure are IgG1.

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

The term "Fd fragment" generally comprises a heavy chain variable region and a constant region CH1; the term "Fv fragment" contains an antibody heavy chain variable region and a light chain variable region, but no constant region, and is the smallest antibody fragment with a complete antigen binding site.

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless 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 include VL-linker-VH or may include VH-linker-VL.

The term "fusion protein" refers to different polypeptides/proteins connected together by genetic recombination or chemical methods to form a larger molecule, which may or may not be connected using a linker.

The term "monoclonal antibody" (or "mAb") refers to an antibody that is essentially homogeneous and directed against a specific antigenic epitope and is produced by a single cell clone. 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 a combination of the above techniques.

The term "chimeric antibody" is a construct in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain or chains is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, and fragments of such antibodies. In a narrow sense, a chimeric antibody comprises all or most of the selected murine heavy and light chain variable regions operably linked to human light and heavy chain constant regions. The constant region sequence or variants or derivatives thereof can be operably 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 per se or can be incorporated into the present disclosure.

The term "humanized antibody" is a hybrid immunoglobulin containing a minimal sequence derived from a non-human immunoglobulin, an immunoglobulin chain or its fragment. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the CDR of the receptor are replaced by residues from the CDR of a non-human species (donor antibody) with desired specificity, affinity and performance, such as mice, rats, rabbits or primates. In some cases, the framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In some cases, "back mutations" can be introduced into a humanized antibody, wherein the residues in one or more FRs of the variable region of the receptor human antibody are replaced by corresponding residues from a non-human species donor antibody. Such back mutations can help maintain the appropriate three-dimensional configuration of one or more grafted CDRs and therefore improve affinity and antibody stability. Antibodies from various donor species can be used, and these donor species include but are not limited to mice, rats, rabbits or non-human primates. In addition, a humanized antibody can be included in a receptor antibody or in a new residue not found in a donor antibody, so as to further improve antibody performance.

"KD" or "Kd" as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. In a specific embodiment, it is determined using surface plasmon resonance (SPR) technology in a Biacore® 8K (Cytiva) instrument.

The "nucleic acid molecule" of the present disclosure refers to a DNA molecule or an RNA molecule. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

The "vector" used in this disclosure refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In a specific embodiment, a vector is also referred to as a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.

The terms "nucleic acid molecule encoding", "coding DNA sequence" and "coding DNA" refer to the order of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

The term "N3pGlu Aβ" refers to the amino acid sequence from Pyr to the 42nd position of Aβ after pyroglutamate cyclization of the third Glu in the amino acid sequence of Aβ 1-42 (SEQ ID NO: 1). "N3pGlu Aβ" and "Aβ N3pE-42" refer to the same sequence (SEQ ID NO: 2) and can be used interchangeably herein.

The term "treatment" as used herein refers to clinical intervention in an attempt to change the disease process caused by an individual or a cell, which can be either preventive or intervention in the clinical pathological process. The therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc.

The "diseases or conditions caused by β-amyloid protein" mentioned in the present disclosure refer to diseases or conditions caused by β-amyloid protein 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 of the three, 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), Bevacizumab disease, etc.), various eye diseases caused by β-amyloid protein deposition (e.g., macular degeneration, drusen-related optic neuropathy, glaucoma, cataracts, etc.), etc.

The term "pharmaceutical composition" refers to a preparation or a combination of preparations containing one, two or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered. When the "pharmaceutical composition" is in the form of a combination of separate preparations containing different, two or more active ingredients, it can be administered simultaneously, sequentially, separately or at intervals, with the purpose of exerting the biological activities of multiple active ingredients and being used together to treat diseases.

"Amyloid β peptide diseases" in the present disclosure are diseases pathologically characterized by Aβ deposition in the brain or in cerebral blood vessels. This includes diseases such as Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy.

Example

The present disclosure is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods in the following examples without specifying specific conditions are generally based on conventional conditions known in the art or conditions recommended by the manufacturer.

Example 1: Engineering of anti-N3pGlu Aβ antibodies

1. Phage library construction

The nucleic acid sequence encoding the light chain variable region sequence VL (SEQ ID NO: 20) of the Remternetug antibody (amino acid sequence information is derived from CN110582511B) was assembled into the pCANTAB-5E-Fab plasmid by molecular cloning to obtain the RemVL-pCANTAB-5E-Fab plasmid carrying the light chain VL encoding nucleic acid sequence. All oligonucleotide primers required for amplifying the nucleic acid sequence encoding VH (SEQ ID NO: 19) of Remternetug were synthesized. For the primers involving the sequence EGGSGSYYNG in HCDR3, NNK (N= A/C/G/T, K= G/T) saturation mutations were introduced at the positions of three deoxyribonucleotides. The primers with mutations (a total of 120) were used to synthesize VH genes by the Assembly PCR method. Each sequence had three NNK mutation sites. The VH encoding nucleic acid sequence with saturation mutation points was then connected to the RemVL-pCANTAB-5E-Fab vector by restriction endonucleases and T4 DNA ligase. The ligation product after desalting was electroporated into SS320 Escherichia coli (manufactured by Lucigen, catalog number 60512-1), and a saturation mutation library with a library capacity of 4.17E7 was constructed. 50 monoclonal clones were randomly selected for sequencing to detect the assembly accuracy.

2. Phage screening

The constructed saturation mutation library was cultured, assisted phage infection, PEG/NaCl precipitation, PBS resuspension and other steps to obtain the packaged phage library. The library was subjected to two rounds of solid panning based on streptavidin magnetic beads. A final concentration of 100μM Aβ 1-42 was added before each round of screening to block the antibody from binding to Aβ 1-42. Aβ pE3-16 (synthesized by Shanghai Jier Biochemical, amino acid sequence Pyr-FRHDSGYEVHHQK-Biotin) with a final concentration of 50nM and 5nM was added in the first and second rounds of screening, respectively. After two rounds of screening, positive clones were selected by phage ELISA and Sanger sequencing was performed to obtain the antibody sequence.

Example 2. Expression and purification of engineered anti-N3pGlu Aβ antibodies

1. Molecular construction

For molecules that are positive for phage ELISA, primers were designed to construct nucleic acid fragments encoding each antibody VH and VL through PCR, and then homologous recombination was carried out with the expression vector pcDNA3.4 (with signal peptide and constant region gene (CH1-Fc/CL) fragment) to construct expression plasmids containing nucleic acids encoding the antibody heavy chain and light chain, respectively. Plasmid preparation was performed after sequencing verification.

2. Expression and purification of humanized antibodies

The antibody was expressed using the ExpiCHO™ expression system (manufactured by Thermo Scientific, catalog number A29133). The plasmids encoding the heavy and light chains of the antibody were transfected into ExpiCHO-S cells at a ratio of 1:1.5. The expression supernatant was collected 8 days after transfection, and impurities were removed by high-speed centrifugation. The antibody was purified using Protein A magnetic beads (manufactured by Nanjing GenScript Technology Co., Ltd., catalog number L00695). The target antibody was eluted with 20mM acetic acid and neutralized with 1M Tris HCl (pH8.0) to obtain the target antibody. After the eluted sample was appropriately concentrated, the solution was changed into PBS buffer and aliquoted for standby use. The final purified antibody was subjected to SDS-PAGE purity analysis, SEC-HPLC purity analysis and A280 concentration determination.

Example 3. Binding affinity to soluble N3pGlu Aβ

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

The affinity of the antibody to soluble N3pGlu Aβ was detected by ELISA to screen for high-affinity anti-N3pGlu Aβ antibodies. After dissolving 1 mg of N3pGlu Aβ polypeptide in 400 μL HFIP (manufacturer Anaiji Chemical, catalog number 920-66-1), the solution was evaporated overnight at room temperature. After evaporation, 1 mL of DMSO was added to fully dissolve it and then used after aliquoting. Take the soluble N3pGlu Aβ dissolved in the above DMSO, add 20 times the volume of PBS buffer, and sonicate to fully dissolve N3pGlu Aβ. Subsequently, the conventional ELISA detection method was used to plate the treated N3pGlu Aβ at 0.2 μg per well (ELISA plate manufacturer is Thermoscientific, catalog number 446469), and incubate at room temperature for 2 hours. After incubation, wash 4 times with PBST (PBS buffer containing 0.5% Tween 20), add blocking solution (PBS buffer containing 3% BSA) and block at room temperature for 2 hours. After blocking, wash with PBST 4 times, add different concentrations of the test antibody, and incubate at room temperature for 1 hour. After incubation, wash with PBST 4 times, add HRP-labeled anti-human Fc secondary antibody (manufacturer: Nanjing GenScript Technology Co., Ltd., catalog number A01854) and incubate for 1 hour. Then wash with PBST 5 times, add 100 μL of TMB colorimetric solution (manufacturer: Cell Signaling Technology, catalog number 7004P6) for color development in the dark for 15 minutes, then add 100 μL of 1M H2SO4 to terminate the reaction, and use an enzyme reader to detect the absorbance at 450 nm.

The experimental results are shown in Table 1 and Figure 1. The anti-N3pGlu Aβ antibodies 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: 1 NO:3, SEQ ID NO:4 and SEQ ID NO:7, 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, the light chain sequence is SEQ ID NO:18) had a significantly higher binding affinity to soluble N3pGlu Aβ polypeptide than Remternetug antibody, and the binding affinity of RemH1, RemH3 and RemH16 to soluble N3pGlu Aβ polypeptide was 22.5 times, 23.2 times and 29.9 times that of Remternetug antibody, respectively.

Table 1. Binding affinity of antibodies to soluble N3pGlu Aβ

B. Surface plasmon resonance technology to detect the binding and dissociation kinetics of the test antibody

The binding dissociation kinetics and affinity of the test antibody to the soluble N3pGlu Aβ peptide were measured by surface plasmon resonance using Biacore® 8K (manufactured by Cytiva). Approximately 8000 RU of the test antibody was first immobilized on the Biacore® CM5 chip by amino coupling, and then the soluble N3pGlu Aβ peptide was diluted 2-fold from 500 nM to 15.6 nM to measure the binding kinetics and affinity. 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 through the reference channel and the experimental channel at 30 μL/min, binding for 3 min, and then dissociating for 10 min. 50mM hydrochloric acid was injected at a flow rate of 30μL/min to regenerate the chip surface for 30s, and regenerated twice. After double subtraction of the data (i.e., the experimental channel signal subtracted the reference channel signal in each cycle, and the sample signal subtracted the blank signal), kinetic fitting was performed using Biacore Insight Evaluation software, and the fitting model used was a 1:1 binding model to obtain kon, koff, and calculate the KD value.

The experimental results are shown in Table 2 below. The screened anti-N3pGlu Aβ antibodies RemH1, RemH3 and RemH16 have significantly higher binding affinity to soluble N3pGlu Aβ polypeptide than Remternetug antibody. The binding affinity of RemH1, RemH3 and RemH16 to soluble N3pGlu Aβ polypeptide is 2.94 times, 37.5 times and 5.07 times that of Remternetug antibody, respectively.

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

Example 4. Binding affinity to aggregated N3pGlu Aβ

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

Then, the binding affinity of the antibody to aggregated N3pGlu Aβ was detected using ELISA method. The specific detection method was the same as that in Part A of Example 3.

The experimental results are shown in Table 3 and Figure 2 below. The binding forces of anti-N3pGlu Aβ antibodies RemH1, RemH3 and RemH16 to aggregated N3pGlu Aβ polypeptides are also significantly improved compared to Remternetug antibodies. The binding forces of RemH1, RemH3 and RemH16 to aggregated N3pGlu Aβ polypeptides are 8.00 times, 5.66 times and 6.49 times that of Remternetug antibodies, respectively.

Table 3. Binding affinity of antibodies to aggregated N3pGlu Aβ

Example 5: Anti-N3pGlu Aβ antibody promotes phagocytosis of Aβ aggregates by BV2 cells

BV2 cells in the logarithmic growth phase (manufacturer: Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60922) were evenly plated in a 24-well plate at 1.0×105 cells per well and cultured overnight in a cell culture incubator at 37°C and 5% CO2. After 24 hours, the anti-N3pGlu Aβ antibody to be tested and the negative control (RSV antibody with no relevant target as negative control) were diluted with DMEM medium, mixed evenly with 10μM Alexa Flour 488-labeled antigen (100% AβN3pE-42 aggregates or 15% AβN3pE-42+85% Aβ1-42 aggregates), and incubated in a 37°C cell culture incubator for 1 hour. The supernatant was then discarded, and the cells were gently washed three times with pre-cooled PBS buffer, and then pre-cooled trypsin was added for digestion for 20 minutes. The digestion was terminated with DMEM medium containing 10% FBS, and then the cells were washed three times with pre-cooled PBS and collected for flow cytometry analysis.

The experimental results are shown in Figure 3a and Figure 3b. The anti-N3pGlu Aβ antibodies RemH1, RemH3, and RemH16 can effectively promote the phagocytosis of antigens by BV2 cells, and the phagocytic ability of cells is significantly stronger than that of Remternetug. And with the increase of antibody concentration, the percentage of antibody-mediated phagocytic cells increases, showing a certain dose dependence.

Example 6: Thermal stability test

A. Differential Scanning Calorimetry (DSC) Analysis

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 changes in the heat capacity of samples during a controlled heating process in real time, obtain thermodynamic characterization parameters of the thermal stability of samples, and consider the higher-order structure and thermal stability of protein drugs.

A differential scanning calorimeter was used to detect the thermal stability of the anti-N3pGlu Aβ antibody disclosed herein. The scanning temperature was from 20°C to 100°C, and the temperature scanning rate was 90°C/hr. The sample to be tested was taken and diluted with a blank buffer (PBS buffer) to a concentration of 1.0 mg/mL of the antibody to be tested, and the blank buffer was used as a blank control for measurement. The Tm value of the antibody to be tested was obtained by software analysis and calculation.

The experimental results are shown in Table 4 below, where the Tm values of RemH3 and RemH16 did not change significantly compared with Remternetug, while the Tm1 value of RemH1 was significantly increased.

Table 4. Tm values of the tested antibodies

B. Thermal stability under stress conditions

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 replaced with PBS buffer, the antibody concentration was adjusted to 10 mg/mL, the sample was sealed and incubated at 65°C for 2 hours, and then immediately placed on ice for cooling. Protein aggregates were removed by centrifugation at 1000 rpm for 5 minutes, and the supernatant solution was transferred to a new EP tube for concentration determination. The unstressed sample was used as a reference, the antigen-antibody binding activity was detected by ELISA, and the percentage of remaining activity after stress was calculated.

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

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

Example 7: Stability test under physiological conditions

In order to further simulate the performance of the test antibody under normal physiological conditions, the antibody solution was replaced with PBS buffer (pH=7.4) and placed at 37°C for 2 weeks (14 days) and 4 weeks (28 days). iCIEF was used to detect the antibody charge distribution after 2 weeks and 4 weeks to determine whether the antibody was stable under normal physiological conditions.

The experimental results are shown in Table 6 below. After the anti-N3pGlu Aβ antibodies RemH1 and RemH16 were placed at 37°C for 2 weeks and 4 weeks, the ratio of the antibody acid region peak was significantly lower than that of Remternetug, and the ratio of the main peak was significantly higher than that of Remternetug. This further shows that the stability of the RemH1 and RemH16 antibodies invented in this study under physiological conditions is significantly stronger than that of the Remternetug antibody.

Table 6. Charge distribution of antibodies after being placed under physiological conditions for different time periods

Example 8. Nonspecific Binding

A. Nonspecific binding to proteins, DNA, and polysaccharides

In order to detect the specificity of the anti-N3pGlu Aβ antibody and verify its non-specific binding to the protein, the ELISA method was used for determination. First, 20 μg of the substance to be tested was coated overnight at 4°C. The coated substance can be fibrinogen (manufactured by Sigma, catalog number F3879), human serum albumin (manufactured by Sigma, catalog number A9511), fibronectin (manufactured by Sigma, catalog number F2006) and low-density lipoprotein (manufactured by Yeasen, catalog number 20613ES05), lipopolysaccharide (manufactured by Thermo Scientific, catalog number 00-4976-93) and double-stranded DNA (manufactured by Sigma, catalog number D4522). Then, PBST was used for washing 4 times. Then, the blocking solution was used for blocking at room temperature for 1 hour, followed by washing 4 times with PBST. After washing, 100 nM of the antibody solution to be tested was added to each well, incubated at room temperature for 2 hours, and then washed 4 times with PBST. Then add the secondary antibody of anti-human Fc-HRP and incubate at room temperature for 1 hour. Then wash with PBST 5 times, add 100 μL TMB colorimetric solution (manufacturer: Cell Signaling Technology, catalog number 7004P6) for 15 minutes in the dark, then add 100 μL 1MH2SO4 to terminate the reaction, and use a microplate reader to detect the absorbance at 450 nm.

The experimental results are shown in Figures 4a and 4b. The anti-N3pGlu Aβ antibodies screened in the present disclosure have no increase in the nonspecificity of RemH3 to fibrinogen, while the nonspecificity of other molecules including RemH1 and RemH16 has no increase, and the specificity effect is not lower than Remternetug.

B. Nonspecific binding to cells

In order to detect the non-specific binding of the anti-N3pGlu Aβ antibody disclosed in the present invention to cells, suspended CHO-S cells and HEK293F cells were used as target cells. First, the concentration of CHO-S cells and HEK293F cells was adjusted to 4×106 cells/mL, and the antibody to be tested (final concentration of 100 nM) and 2×105 target cells were added to a 96-well plate with a total volume of 100 μL, and incubated at 4°C for half an hour. After the incubation is completed, the cells were washed three times with FACS buffer (PBS buffer containing 2% FBS) pre-cooled at 4°C, and then anti-Human Fc secondary antibody with PE fluorescence (manufacturer Biolegend, catalog number 410708) was added and incubated at 4°C for half an hour. After the incubation is completed, the cells were washed three times with FACS buffer pre-cooled at 4°C, and finally resuspended with FACS buffer, the cell concentration was adjusted, the cell fluorescence intensity was detected by flow cytometry, and the data was sorted and analyzed.

The experimental results are shown in Figure 5. In the non-specific binding experiment with CHO-S cells, the anti-N3pGlu Aβ antibodies RemH1, RemH3, and RemH16 obtained by the present disclosure screening did not increase the non-specificity compared with Remternetug. In the non-specific binding experiment with HEK293F cells, the non-specificity of RemH16 molecules was similar to that of Remternetug, and the non-specificity of RemH1 and RemH3 molecules was slightly increased.

sequence

SEQ ID NO: 1 Aβ1-42

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

SEQ ID NO: 2 Aβ N3pE-42

Pyr-FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

SEQ ID NO: 3 RemH1, 3, 16 HCDR1

SYPMS

SEQ ID NO: 4 RemH1, 3, 16 HCDR2

AISGSGGSTYYADSVKG

SEQ ID NO: 5 RemH1 HCDR3

EGGIGSIYEGFDY

SEQ ID NO: 6 RemH3 HCDR3

EGGSGPYFRGFDY

SEQ ID NO: 7 RemH16 HCDR3

EGGSGPYFGGFDY

SEQ ID NO: 8 RemH1, 3, 16 LCDR1

RASQSLGNWLA

SEQ ID NO: 9 RemH1, 3, 16 LCDR2

QASTLES

SEQ ID NO: 10 RemH1, 3, 16 LCDR3

QHYKGSFWT

SEQ ID NO: 11 RemH1 VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGIGSIYEGFDYWGQGTLVTVSS

SEQ ID NO: 12 RemH3 VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGPYFRGFDYWGQGTLVTVSS

SEQ ID NO: 13 RemH16 VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGPYFGGFDYWGQGTLVTVSS

SEQ ID NO: 14 RemH1, 3, 16 VL

DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK

SEQ ID NO: 15 RemH1 Heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGIGSIYEGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 16 RemH3 Heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGPYFRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 17 RemH16 Heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGPYFGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 18 RemH1, 3, 16 Light Chian

DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

SEQ ID NO:19 Remternetug(201c)VH

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYNGFDYWGQGTLVTVSS

SEQ ID NO:20 Remternetug(201c)VL

DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK

Claims (17)

1. An antibody that binds to N3pGlu Aβ, wherein the antibody can specifically bind 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, and the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are respectively: SEQ ID NO: 3, 4, 7, 8, 9, 10. 2. 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 and the light chain variable region have the following amino acid sequences, respectively: SEQ ID NO:13 and 14. 3. 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 a HCDR region sequence of a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 13, and the light chain variable region comprises a LCDR sequence of a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 14, wherein the HCDR or LCDR sequence is determined according to the Chothia, IMGT or AHo numbering system. 4. The antibody binding to N3pGlu Aβ according to any one of claims 1 to 3, further having one or more of the following characteristics: (1) It also includes a heavy chain constant region and/or a light chain constant region; (2) It is a mouse 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 one of the fused parts comprises the antibody binding to N3pGluAβ according to any one of claims 1 to 4. 6. A bispecific antibody or a multispecific antibody, wherein one of the antigen-binding domains comprises the antibody that binds to N3pGlu Aβ according to any one of claims 1 to 4. 7. A nucleic acid encoding the antibody binding to N3pGlu Aβ according to any one of claims 1 to 4. 8. A recombinant vector comprising the nucleic acid of claim 7. 9. A host cell comprising the recombinant vector of claim 8 or the nucleic acid of claim 7. 10. The host cell of claim 9, which is selected from a prokaryotic cell or a eukaryotic cell. The host cell according to claim 10 , wherein the prokaryotic cell is Escherichia coli and the eukaryotic cell is yeast or mammalian cell. 12. The host cell of claim 11, wherein the mammalian cell is selected from CHO cells, NSO cells, Expi293 cells or HEK293 cells. 13. A method for preparing the antibody binding to N3pGlu Aβ according to any one of claims 1 to 4, comprising: culturing the host cell according to any one of claims 9 to 12 under suitable conditions, and purifying the expression product from the cell. 14. A pharmaceutical composition comprising the antibody binding to N3pGlu Aβ according to any one of claims 1 to 4, or the fusion protein according to claim 5, or the bispecific antibody or multispecific antibody according to claim 6, or the nucleic acid according to claim 7, or the recombinant vector according to claim 8, or the host cell according to any one of claims 9 to 12. 15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier. 16. The pharmaceutical composition of claim 15, further comprising an additional other therapeutic agent. 17. Use of the antibody binding to N3pGlu Aβ according to any one of claims 1 to 4, the fusion protein according to claim 5, the bispecific antibody or multispecific antibody according to claim 6, the nucleic acid according to claim 7, the recombinant vector according to claim 8, the host cell according to any one of claims 9 to 12, or the pharmaceutical composition according to any one of claims 14 to 16 in the preparation of a medicament for preventing, delaying and/or treating a disease, wherein the disease is selected from Alzheimer's disease (AD), Down syndrome or cerebral amyloid angiopathy (CAA).