Anti-aβ1-42 antibody, preparation method therefor, and use thereof

Abstract

The present application provides a method for preparing a humanized Aβ1-42 antibody and use thereof. Specifically, the antibody of the present application combines a murine antibody variable region and a human antibody framework region; on the basis of specifically binding to an Aβ peptide segment, the antibody reduces immunogenicity due to its humanized framework region. The antibody has the advantages of high affinity, high specificity, low immunogenicity, and scalability for large-scale production. The antibody of the present application can be used for detecting an Aβ1-42 antigen, eliminating Aβ deposits, or treating related diseases thereof, such as Alzheimer's disease.

Description

Anti-aβ1-42 antibody and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological immunology, and specifically relates to an anti-aβ1-42 antibody and a preparation method and application thereof. BACKGROUND

[0002] In the field of biotechnology, pharmaceutical research and development, and immunology, the development of antibodies is an important research direction. Alzheimer's disease is a chronic neurodegenerative disease that mainly affects memory, thinking and behavior in the brain. Its pathological features include the formation of amyloid beta (Aβ) plaques, tau protein tangles and neuronal loss. Among them, Aβ1-42 is a specific form of Aβ, which plays a key role in the pathological process of AD. Studies have shown that the abnormal accumulation of Aβ1-42 may be the main cause of triggering a series of downstream events leading to nerve cell damage and death.

[0003] Although existing technologies can obtain antibodies against Aβ1-42, there are some problems and limitations. First, since these antibodies are obtained from animals, they may have incompatibility with the human immune system, i.e. so-called "xenoprotein reaction". This reaction can lead to reduced efficacy of the antibody or induce adverse reactions. Second, existing technologies still face challenges in obtaining antibodies with high affinity and high specificity, and some antibodies still lack specificity in targeting Aβ1-42 or have certain binding with Aβ1-40, which affects the clinical application of the antibodies. In addition, existing technologies also have bottlenecks in the large-scale production of antibodies, which limits the wide application of the antibodies.

[0004] Therefore, there is an urgent need in the art to develop Aβ1-42 antibodies that are humanized, high affinity, high specificity and easy to scale up. SUMMARY

[0005] The present application provides an anti-aβ1-42 antibody that is humanized, high affinity, high specificity and easy to scale up. The present application also provides antibodies, chimeric antigen receptors, fusion proteins, recombinant proteins based on the anti-aβ1-42 antibody, and their encoding nucleic acids, expression vectors, host cells, immunoconjugates, etc., and provides pharmaceutical compositions comprising the above active ingredients.

[0006] In the first aspect of the present application, an anti-aβ1-42 antibody or an antibody binding fragment thereof is provided, which comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0007] (1) The heavy chain variable region comprises the following three CDRs as determined according to the Kabat rule: VH-CDR1 shown in SEQ ID NO:11, VH-CDR2 shown in SEQ ID NO:12, and VH-CDR3 shown in SEQ ID NO:13; and / or

[0008] The light chain variable region comprises the following three CDRs as determined according to the Kabat rule: VL-CDR1 shown in SEQ ID NO:14, VL-CDR2 shown in SEQ ID NO:15, and VL-CDR3 shown in SEQ ID NO:16; or

[0009] (2) The heavy chain variable region comprises the following three CDRs as determined according to the IMGT rules: VH-CDR1 shown in SEQ ID NO:17, VH-CDR2 shown in SEQ ID NO:18, and VH-CDR3 shown in SEQ ID NO:19; and / or

[0010] The light chain variable region, as determined according to the IMGT rule, includes the following three CDRs: VL-CDR1 shown in SEQ ID NO:20, VL-CDR2 shown in SEQ ID NO:21, and VL-CDR3 shown in SEQ ID NO:22.

[0011] In another preferred embodiment, the antibody or its antibody-binding fragment is murine, humanized, or chimeric.

[0012] In another preferred embodiment, the heavy chain variable region (VH) is selected from the group consisting of:

[0013] (H1) Heavy chain variable region as shown in SEQ ID NO:3; or

[0014] (H2) Heavy chain variable region as shown in SEQ ID NO:4; or

[0015] (H3) Heavy chain variable region as shown in SEQ ID NO:9.

[0016] In another preferred embodiment, the light chain variable region (VL) is selected from the group consisting of:

[0017] (L1) Light chain variable region as shown in SEQ ID NO:1; or

[0018] (L2) Light chain variable region as shown in SEQ ID NO:2; or

[0019] (L3) Light chain variable region as shown in SEQ ID NO:10.

[0020] In another preferred embodiment, the heavy chain variable region (VH) of the antibody or its antibody-binding fragment is: (H2) the heavy chain variable region as shown in SEQ ID NO:4; and the light chain variable region (VL) is: (L1) the light chain variable region as shown in SEQ ID NO:1.

[0021] In another preferred embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4 or SEQ ID NO:3; and / or

[0022] The antibody or antigen-binding fragment comprises a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:2.

[0023] In another preferred embodiment, the antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the three heavy chain CDRs and a human heavy chain framework region for connecting the heavy chain CDRs; and the light chain variable region includes the three light chain CDRs and a human light chain framework region for connecting the light chain CDRs.

[0024] In another preferred embodiment, the humanized Aβ1-42 antibody or its antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody (“scFv”), bisscFv, (scFv)2, microantibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein (“dsFv”), or combinations thereof.

[0025] In another preferred embodiment, the antibody specifically binds to Aβ peptides 1-42 but not to Aβ peptides 1-40.

[0026] In another preferred embodiment, the antibody specifically binds to epitopes at positions 37-42 of the Aβ peptide 1-42.

[0027] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0028] In another preferred embodiment, the antibody is a monoclonal antibody.

[0029] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies.

[0030] A second aspect of this application provides a recombinant protein, said recombinant protein comprising:

[0031] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of this application; and

[0032] (ii) Optional tag sequences to assist in expression and / or purification.

[0033] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS (SEQ ID NO:23) sequence, and a FLAG tag.

[0034] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0035] In another preferred embodiment, the recombinant protein is a fusion protein.

[0036] In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against Aβ1-42), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0037] In another preferred embodiment, the bispecific or multispecific antibody not only targets Aβ1-42 but also specifically binds to additional target antigens (such as other Alzheimer's disease antigens).

[0038] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0039] In another preferred embodiment, the recombinant protein further includes an additional fusion element (or fusion polypeptide fragment) fused together with said element (i).

[0040] In a third aspect, this application provides a chimeric antigen receptor (CAR) whose antigen-binding domain contains an antibody single-chain variable region sequence scFv targeting Aβ1-42, wherein the heavy chain variable region and light chain variable region contained in the scFv are as defined in the first aspect of this application, or the heavy chain sequence and light chain sequence contained in the scFv are as defined in the first aspect of this application.

[0041] In a fourth aspect, this application provides a polynucleotide that encodes an antibody or antigen-binding fragment thereof as described in the first aspect of this application, a recombinant protein as described in the second aspect of this application, or a chimeric antigen receptor as described in the third aspect of this application.

[0042] In a fifth aspect, this application provides a vector containing the polynucleotide described in the fourth aspect of this application.

[0043] In another preferred embodiment, the vector is selected from the group consisting of plasmids, viruses (such as lentiviruses, adenoviruses, AAV viruses, retroviruses), granules, or combinations thereof.

[0044] In another preferred embodiment, the vector is a plasmid, preferably an expression vector.

[0045] In another preferred embodiment, the vector is a viral vector.

[0046] In a sixth aspect of this application, a host cell is provided, wherein the host cell contains the vector described in the fifth aspect of this application or wherein the exogenous polynucleotide described in the fourth aspect of this application is integrated into its genome.

[0047] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0048] In another preferred embodiment, the cell is a somatic cell.

[0049] In another preferred embodiment, the cell is a mammalian cell.

[0050] In another preferred embodiment, the cell is an immune cell (such as an NK cell or a T cell).

[0051] In another preferred embodiment, the host cell is an engineered cell.

[0052] In a seventh aspect, this application provides a CAR-NK cell or CAR-T cell or a method for preparing the same, wherein the CAR-NK cell or CAR-T cell expresses the chimeric antigen receptor described in the third aspect of this application.

[0053] In another preferred embodiment, the preparation method includes the following steps:

[0054] The polynucleotide described in the fourth aspect of this application or the vector described in the fifth aspect of this application is introduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

[0055] An eighth aspect of this application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect of this application, a recombinant protein as described in the second aspect of this application, a chimeric antigen receptor as described in the third aspect of this application, a polynucleotide as described in the fourth aspect of this application, a carrier as described in the fifth aspect of this application, or a host cell as described in the sixth aspect of this application, or a combination thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0056] In another preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

[0057] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0058] In another preferred embodiment, the pharmaceutical composition comprises 0.01-99.99% of an antibody or antigen-binding fragment thereof as described in the first aspect of this application, a recombinant protein as described in the second aspect of this application, a chimeric antigen receptor as described in the third aspect of this application, a polynucleotide as described in the fourth aspect of this application, a carrier as described in the fifth aspect of this application, a host cell as described in the sixth aspect of this application, or a combination thereof, and 0.01-99.99% of a pharmaceutical carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0059] In another preferred embodiment, the pharmaceutical composition is used to treat Alzheimer's disease or to clear the Aβ peptide Aβ1-42.

[0060] A ninth aspect of this application provides an immunoconjugate comprising:

[0061] (a) An antibody portion, said antibody portion being selected from the group consisting of: antibodies or antigen-binding fragments thereof as described in the first aspect of this application, recombinant proteins as described in the second aspect of this application, or combinations thereof; and

[0062] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0063] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0064] A tenth aspect of this application provides the use of an antibody or antigen-binding fragment thereof as described in the first aspect of this application, a recombinant protein as described in the second aspect of this application, a chimeric antigen receptor as described in the third aspect of this application, a polynucleotide as described in the fourth aspect of this application, a vector as described in the fifth aspect of this application, a host cell as described in the sixth aspect of this application, a CAR-NK cell or CAR-T cell as described in the seventh aspect of this application, a pharmaceutical composition as described in the eighth aspect of this application, or an immunoconjugate as described in the ninth aspect of this application, for the preparation of a drug or formulation, wherein the drug or formulation is used for:

[0065] (a) Treatment of AD; and / or

[0066] (b) Clear Aβ plaques from the brain;

[0067] (c) Detect the presence of Aβ in the sample.

[0068] In another preferred embodiment, the drug is used in combination with other drugs that target different epitopes of Aβ.

[0069] In the eleventh aspect of this application, a method for preparing an anti-Aβ1-42 antibody is provided, the method comprising:

[0070] (a) Culturing the host cells described in the sixth aspect of this application under suitable expression conditions;

[0071] (b) Isolating an antibody from a culture, wherein the antibody is the antibody or antigen-binding fragment thereof described in the first aspect of this application.

[0072] In a twelfth aspect of this application, a kit for detecting the Aβ1-42 peptide is provided, the kit comprising an antibody or antigen-binding fragment thereof as described in the first aspect of this application as a first detection reagent.

[0073] In another preferred embodiment, the kit further includes an instruction manual describing a method for detecting Aβ1-42 protein.

[0074] In another preferred embodiment, the kit comprises:

[0075] A first container, and an antibody or antigen-binding fragment thereof as described in the first aspect of this application located in the first container;

[0076] The second container, and the second detection reagent located in the second container, which targets and binds to regions other than the Aβ1-42 peptide of the Aβ protein.

[0077] In another preferred embodiment, the second detection reagent is an antibody or its antigen-binding fragment.

[0078] In another preferred embodiment, the kit further includes a third container and an antibody or antigen-binding fragment thereof targeting and binding to the Aβ1-40 peptide located in the third container.

[0079] In a thirteenth aspect of this application, a method for detecting the Aβ peptide Aβ1-42 is provided, the method comprising the steps of:

[0080] a) Contact the sample to be tested with the antibody described in the first aspect of this application; and

[0081] b) Detect whether an "antigen Aβ1-42-antibody complex" (or a first antigen-antibody complex) is generated.

[0082] The formation of the “antigen Aβ1-42-antibody complex” indicates the presence of the Aβ peptide Aβ1-42 in the sample.

[0083] In another preferred embodiment, the method further includes:

[0084] The sample to be tested is contacted with the antibody described in this application and other Aβ antibodies targeting sites other than 1-42 of the Aβ protein, and the formation of a second antigen-antibody complex is detected.

[0085] In another preferred embodiment, the sample to be tested is derived from the subject's blood, cerebrospinal fluid, or brain tissue.

[0086] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0087] In another preferred embodiment, the formation of an antigen-antibody complex indicates the presence of Aβ1-42 protein in the sample to be tested.

[0088] In the fourteenth aspect of this application, a method for preparing a humanized Aβ1-42 antibody is provided.

[0089] (a) The peptide at positions 37-42 of Aβ amyloid protein was conjugated to a carrier protein to obtain the conjugated carrier protein. The conjugated carrier protein was then used as an antigen to immunize mice, thereby obtaining mouse antibodies against Aβ1-42; and

[0090] (b) Humanize the non-CDR region of the mouse antibody against Aβ1-42 to obtain a humanized anti-Aβ1-42 antibody.

[0091] In another preferred embodiment, in step (a), GGC is added to the C-terminus of the polypeptide at positions 37-42, and then it is coupled with the carrier protein.

[0092] In another preferred embodiment, the carrier protein is selected from the group consisting of serum albumin (BSA), keyhole hemocyanin (KLH), or combinations thereof.

[0093] In another preferred embodiment, the method includes the following steps:

[0094] S1: Preparation of mouse Aβ1-42 antibody;

[0095] S2: Screen for high-affinity antibodies and obtain antibody sequences;

[0096] S3: Humanize the obtained antibody sequences using computer software;

[0097] Step S1 includes: extracting positions 37-42 of Aβ amyloid protein as the Aβ antigen region for polypeptide synthesis, adding GGC-conjugated carrier protein to the C-terminus during synthesis; immunizing mice with the antigen; fusing mouse spleen cells with myeloma cells using electrofusion technology to form fusion cells; and using HAT to screen for high-purity fusion cells.

[0098] Step S2 includes: combining recombinant Aβ1-42 antigen with high-purity fusion cells, identifying positive cells by ELISA, wherein the positive cells are a cell population with an OD450 value greater than 3 times that of the negative control as measured by an enzyme-linked immunosorbent assay (ELISA), and the positive cells can express high-affinity antibodies; performing monoclonalization on the positive cells twice, and identifying each monoclonalization by ELISA; finally obtaining a monoclonal hybridoma cell line, and obtaining the antibody variable region sequence using hybridoma sequencing.

[0099] Step S3 includes: comparing the heavy and light chain variable region germline gene databases of the IMGT human antibody to select germline genes of the heavy and light chain variable regions with high homology to the chimeric antibody variable region as templates; and using the Kabat numbering system to determine the amino acid number and CDR region of the antibody. The CDRs of the chimeric antibody are then transplanted into the frame region (FR) of the corresponding human template, forming a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Multiple factors are quantified to score the suitability of the template, and the transplanted sequence of the high-scoring template is selected as the basis for reversion mutation design. These multiple factors include: homology, frequency of occurrence in clinical and human trials, historical expression data, isoelectric point of the transplanted sequence, and high-risk PTM sites.

[0100] In the fifteenth aspect of this application, a method of treating a disease associated with Aβ plaque deposition is provided, comprising administering to a subject in need of treatment an appropriate amount of an antibody or antigen-binding fragment thereof as described in the first aspect of this application, a recombinant protein as described in the second aspect of this application, a host cell as described in the sixth aspect of this application, or a pharmaceutical composition as described in the eighth aspect of this application.

[0101] In another preferred embodiment, the disease associated with Aβ plaque deposition is Alzheimer's disease.

[0102] It should be understood that, within the scope of this application, the above-described technical features and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0103] Figure 1 shows the linear fitting results of the ELISA test results. Detailed Implementation

[0104] Through extensive and in-depth research, the inventors have developed a humanized Aβ1-42 antibody. Specifically, this antibody binds to both a murine antibody variable region and a human antibody framework region. While specifically binding to the Aβ peptide, its humanized framework region reduces immunogenicity. Furthermore, the Aβ1-42 antibody of this application does not bind to the Aβ1-40 antigen, exhibiting advantages such as high affinity, high specificity, and low immunogenicity. This application was completed based on these findings.

[0105] the term

[0106] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Before describing this application, it should be understood that this application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary.

[0107] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0108] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included in this application. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered in this application, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0109] As used herein, the term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0110] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0111] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[0112] As used herein, the term "antigen-binding domain" and the like encompasses any naturally occurring, enzymatically available, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains, can be used, for example, deriving antigen-binding fragments of antibodies from intact antibody molecules. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, such as to arrange one or more variable domains and / or constant domains into a suitable layout, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0113] As used herein, non-limiting examples of antigen-binding fragments or antigen-binding domains include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., independent complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0114] As used herein, antigen-binding fragments or antigen-binding domains will generally contain at least one variable domain. The variable domain can have any size or amino acid composition and will typically contain at least one CDR adjacent to or conforming to one or more frame sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be arranged opposite each other in any suitable configuration. For example, the variable region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers. Optionally, the antigen-binding domain can contain monomeric VH or VL domains.

[0115] In a given antibody's light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia based on the antibody's three-dimensional structure and the topology of the CDR loops; Kabat (Kabat, E., et al., USDapartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); the International Immuno GeneTics database (IMGT); the EU numbering system; and Chothia definitions based on loop structural positions.

[0116] It should be understood that the precise amino acid sequence boundaries of the CDR in this application can optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise stated, in this application, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system.

[0117] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0118] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0119] As those skilled in the art know, immunoconjugates and fusion expression products include conjugates formed by combining drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibody or fragment thereof of this application.

[0120] In a preferred embodiment of this application, the heavy chain variable region of the nanobody or antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0121] In a preferred embodiment of this application, the heavy chain of the nanobody or antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0122] This application also provides other proteins or fusion expression products containing the antibody of this application. Specifically, this application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of this application, preferably at least 95% homology.

[0123] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four backbone regions (FR). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of similar antibodies can be compared to determine which amino acids constitute the FR or CDR regions.

[0124] This application includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this application also includes fragments, derivatives, and analogs of said antibodies.

[0125] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the same biological function or activity as the antibody of this application. The polypeptide fragments, derivatives, or analogs of this application may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0126] The term "antibody" in this application refers to a polypeptide having Aβ1-42 binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibody of this application. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this application.

[0127] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of this application under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of this application.

[0128] This application also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to nearly full-length polypeptides, this application also includes fragments of the antibodies of this application. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of this application.

[0129] In this application, "a conserved variant of the antibody of this application" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0130] Table A

[0131] This application also provides a polynucleotide molecule encoding the aforementioned antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of this application may be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0132] The polynucleotides encoding the mature polypeptide of this application include: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0133] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0134] This application also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. This application particularly relates to polynucleotides that hybridize with the polynucleotides described in this application under stringent conditions. In this application, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0135] The full-length nucleotide sequence or fragments of the antibody described in this application can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Generally, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0136] Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this application include biomolecules existing in isolated forms.

[0137] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this application can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this application through chemical synthesis.

[0138] This application also relates to vectors comprising the aforementioned suitable DNA sequence and suitable promoter or control sequence. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0139] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0140] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0141] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this application. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture should be carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0142] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0143] The nanobodies or antibodies of this application can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0144] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0145] Therapeutic agents that can bind to or conjugate with the antibody of this application include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0146] Humanized Aβ1-42 antibody

[0147] As used herein, the terms "Aβ1-42 antibody of this application" and "anti-Aβ1-42 antibody of this application" are used interchangeably and both refer to the antibody described in the first aspect of this application.

[0148] This application includes heavy chain variable regions and light chain variable regions selected from the following group:

[0149] (1) According to Kabat rules: the heavy chain variable region contains the following three CDRs: VH-CDR1 shown in SEQ ID NO:11, VH-CDR2 shown in SEQ ID NO:12, and VH-CDR3 shown in SEQ ID NO:13; and / or

[0150] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:14, VL-CDR2 shown in SEQ ID NO:15, and VL-CDR3 shown in SEQ ID NO:16; or

[0151] (2) According to the IMGT rule: the heavy chain variable region contains the following three CDRs: VH-CDR1 shown in SEQ ID NO:17, VH-CDR2 shown in SEQ ID NO:18, and VH-CDR3 shown in SEQ ID NO:19; and / or

[0152] The light chain variable region includes the following three CDRs: VL-CDR1 shown in SEQ ID NO:20, VL-CDR2 shown in SEQ ID NO:21, and VL-CDR3 shown in SEQ ID NO:22.

[0153] Preferably, the antibody described herein is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0154] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0155] The antibody in this application may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0156] The antibody derivatives described in this application may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0157] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.

[0158] The animal is preferably a mammal, such as a mouse.

[0159] In the above-mentioned content of this application, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0160] In the above-mentioned content of this application, more preferably, the number of added, deleted, modified and / or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0161] Recombinant protein

[0162] This application also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2), and heavy chain CDR3 (VH-CDR3) of the antibody of this application, and / or one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2), and light chain CDR3 (VL-CDR3) of the antibody of this application.

[0163] Preferably, the recombinant protein further includes an antibody heavy chain constant region and / or an antibody light chain constant region. The antibody heavy chain constant region is conventional in the art, preferably a rat-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and more preferably a human-derived antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat-derived antibody light chain constant region or a human-derived antibody light chain constant region, and more preferably a human-derived antibody light chain constant region.

[0164] In a preferred embodiment, the recombinant protein includes the antibody of this application.

[0165] The recombinant protein is a conventional protein in the art, preferably one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies.

[0166] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.

[0167] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, comprising an Fd segment of a light chain variable region, a light chain constant region, and a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0168] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0169] The single-region antibody described is a conventional single-region antibody in the art, which includes only the heavy chain variable region.

[0170] The recombinant protein is prepared using conventional methods in the art. Preferably, the preparation method involves isolating the protein from an expression transformant or obtaining it through artificial synthesis of the protein sequence. The method for isolating the protein from the expression transformant is preferably as follows: cloning a polynucleotide molecule encoding the protein and carrying a point mutation into a recombinant vector; transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant; and culturing the obtained recombinant expression transformant to isolate and purify the recombinant protein.

[0171] Polynucleotides

[0172] This application also provides a polynucleotide encoding the above-described chimeric antigen receptor (CAR) construct of the antibody or recombinant protein of this application or the antibody of this application.

[0173] The preparation method of the polynucleotide is a conventional preparation method in the art. Preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full-sequence synthesis.

[0174] Those skilled in the art will understand that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately modified by substitution, deletion, alteration, insertion, or addition to provide a polynucleotide homologue. The polynucleotide homologue of this application can be prepared by substituting, deleting, or adding one or more bases of the gene encoding the protein sequence, while maintaining antibody activity.

[0175] carrier

[0176] This application also provides a recombinant expression vector containing the said nucleic acid.

[0177] The recombinant expression vector described herein can be obtained using conventional methods in the art, namely, by linking the nucleic acid molecules described in this application to various expression vectors. The expression vectors are any conventional vectors in the art, as long as they can accommodate the aforementioned nucleic acid molecules. Preferably, the vectors include various plasmids, granules, bacteriophages, or viral vectors, etc.

[0178] This application also provides a recombinant expression transformant comprising the above-described recombinant expression vector.

[0179] The recombinant expression transformant is prepared using conventional methods in the art, preferably by transforming the recombinant expression vector into host cells. The host cells can be any type of host cell commonly used in the art, as long as they allow the recombinant expression vector to replicate stably and effectively express the carried nucleic acid. Preferably, the host cells are E. coli TG1 or E. coli BL21 cells (expressing single-chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies). Transforming the aforementioned recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of this application. The transformation method is a conventional method in the art, preferably chemical transformation, heat shock, or electroporation.

[0180] Antibody preparation

[0181] The DNA sequence of the antibody or fragment thereof in this application can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single-chain antibody.

[0182] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0183] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0184] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of this application can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this application through chemical synthesis.

[0185] This application also relates to vectors comprising the aforementioned suitable DNA sequence and suitable promoter or control sequence. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0186] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0187] Typically, host cells transformed are cultured under conditions suitable for antibody expression according to this application. The antibody is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography—common separation and purification methods well-known to those skilled in the art—to obtain the antibody of this application.

[0188] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0189] The antibodies of this application can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0190] Antibody-drug conjugates (ADCs)

[0191] As used in this article, the terms “antibody-drug conjugate,” “ADC,” and “antibody-drug conjugate” are used interchangeably.

[0192] This application also provides antibody-drug conjugates (ADCs) based on the antibodies of this application.

[0193] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0194] The antibody and the effector molecule in this application can be coupled via a coupling agent. Examples of such coupling agents include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide-based coupling agents. Non-selective coupling agents refer to compounds that covalently link the effector molecule and the antibody, such as glutaraldehyde. Carboxyl-based coupling agents can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0195] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0196] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0197] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0198] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0199] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0200] In this application, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0201] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0202] This application also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0203] In some embodiments, the method of this application includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of this application further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0204] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0205] in:

[0206] Ab is an antibody.

[0207] LU stands for connector;

[0208] D is a drug;

[0209] Furthermore, the subscript p is a value selected from 1 to 8.

[0210] application

[0211] This application also provides the use of the antibodies, recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of this application, for example, for the preparation of diagnostic agents or pharmaceuticals.

[0212] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with Aβ deposition.

[0213] Preferably, the diagnostic agent is used to treat Alzheimer's disease.

[0214] Reagent test kit

[0215] This application also provides a kit containing the antibody (or fragment thereof) of this application, the kit further including a container, instructions for use, buffer, etc.

[0216] The kit of this application also includes other antibodies or drugs targeting different epitopes of Aβ, which are used in combination with the antibody described in this application to clear Aβ plaques.

[0217] Pharmaceutical Composition

[0218] This application also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0219] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition described in this application is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0220] The antibody described in this application can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0221] The pharmaceutical composition of this application contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or its conjugate) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical composition of this application can be formulated into an injectable form, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions should preferably be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the polypeptide of this application can also be used with other therapeutic agents.

[0222] Preferably, the pharmaceutical composition of this application further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0223] In this application, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the norm.

[0224] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0225] Compared with the prior art, the advantages of this application are as follows:

[0226] 1. This invention addresses the problem of incompatibility between antibodies obtained through existing technologies and the human immune system, known as "heterologous protein reaction." Through humanized design, the immunogenicity of the antibodies is reduced, thereby improving the safety of their clinical application.

[0227] 2. This invention addresses the challenges of obtaining antibodies with high affinity and high specificity using existing technologies. By employing computer software for humanization design, the affinity and specificity of the antibodies are optimized, thereby improving their clinical application effectiveness.

[0228] 3. It has solved the bottleneck problem of existing technologies in the large-scale production of antibodies. Through recombinant expression technology, it has achieved large-scale production of antibodies and met market demand.

[0229] 4. It solves the problem that existing technologies require a large number of experimental animals and a cumbersome screening process. By obtaining antibody sequences through immunization of mice and screening of hybridomas, the antibody research and development process is simplified and the research and development efficiency is improved.

[0230] 5. It addresses the shortcomings of existing technologies in the research and application of antibodies against Aβ1-42, and provides a new tool for the diagnosis and treatment of Alzheimer's disease by preparing humanized Aβ1-42 antibodies.

[0231] 6. The antibody in this application is a humanized antibody optimized through genetic engineering, which retains high affinity for the antigen while enhancing the specificity of epitope recognition. The antibody and its IVD diagnostic kit are highly suitable for detecting low-abundance biomarkers, such as Alzheimer's disease, while reducing non-specific binding to other proteins and improving the signal-to-noise ratio.

[0232] 7. Humanized antibodies are usually expressed using mammalian cells (such as CHO cells), and the production process is standardized, making them suitable for large-scale production with minimal batch-to-batch variation, ensuring the stability of reagent quality. Compared to traditional animal-derived antibodies, humanized antibodies are less prone to degradation during storage, extending the shelf life of the reagents.

[0233] 8. Humanized antibodies have a well-defined Fc region structure, which facilitates efficient conjugation with markers such as enzymes and fluoresceins. They are suitable for various IVD platforms such as ELISA, chemiluminescence, flow cytometry, and immunochromatography, without the need to adjust the process due to differences in antibody sources.

[0234] 9. The humanized antibody of this application significantly improves the accuracy, reliability, and applicability of IVD reagents by optimizing immunogenicity, stability, and specificity, especially in high-precision diagnostics and complex sample testing. With advancements in bioengineering technology, its cost is gradually decreasing, and it is expected to become the mainstream choice for IVD raw materials in the future.

[0235] The present application 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 application. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0236] Example 1. Preparation of mouse Aβ1-42 antibody

[0237] (1) Immunogen preparation

[0238] The Aβ antigen region is extracted from positions 37-42 of human Aβ amyloid protein, with the following sequence:

[0239] MVGGVV (SEQ ID NO:24) was then synthesized into a peptide by adding GGC to the C-terminus to obtain a 9-peptide.

[0240] The 9-peptide was covalently modified to conjugate the carrier protein BSA to its C-terminal carboxyl group. The resulting conjugated protein was used as an immunogen for subsequent animal immunization.

[0241] (2) Antigen Immunization

[0242] Healthy Balb / c mice were selected and immunized with the antigen. Specifically, the immunogen was injected into the mice, and booster immunizations were administered every two weeks for a total of four immunizations. For the first immunization, the immunogen was mixed with an equal volume of Freund's adjuvant to form an emulsion, which was injected subcutaneously at multiple sites. For the second and third immunizations, the antigen was mixed with an equal volume of Freund's adjuvant to form an emulsion, which was also injected subcutaneously at multiple sites. Two booster immunizations were administered. Serum samples were collected after the three immunizations, and serum titers were determined by ELISA. Mice with high serum titers were given a single intraperitoneal injection of 100 μg of the immunogen, followed by spleen harvesting. During this period, the immune response of the mice was closely monitored to ensure that an effective immune response was generated.

[0243] (3) Preparation of SP / 20 cells

[0244] Before antigen immunization is complete, SP / 20 cells, a commonly used myeloma cell line, are resuscitated. SP / 20 cells are cultured in RPMI-1640 medium containing 10% fetal bovine serum. Cell viability is maintained at >95% before fusion, and the cells are washed 2–3 times with serum-free RPMI-1640 medium to remove serum.

[0245] (4) Preparation of mouse spleen cells

[0246] After euthanizing the mice, the spleen tissue was aseptically removed. 10 mL of serum-free culture medium (RPMI 1640) was drawn up using a syringe. Holding forceps in the left hand, the syringe needle was inserted into one end of the spleen in the right hand, and the spleen cells were gently blown out. The spleen showed a lightening of color. The obtained cell suspension was collected into a 50 mL centrifuge tube, ground, and strained. The cell suspension was then collected into the centrifuge tube. The cells were washed twice with serum-free culture medium, counted, and resuspended in serum-free culture medium before being transferred to a centrifuge tube.

[0247] (5) Fusion of mouse spleen cells and myeloma cells

[0248] Mouse spleen cells were electrofused with SP / 20 cells using electrofusion. First, the prepared SP / 20 cells and mouse spleen cells were mixed at a specific ratio, then washed twice with electrofusion buffer, followed by pulsed fusion according to the electrofusion protocol. After fusion, the fused cells were seeded into 96-well plates, and HAT was added for screening 24 hours later. The growth of the fused cells was observed after 5-7 days.

[0249] (6) Antibody screening

[0250] Recombinant Aβ1-42 antigen was coated onto 96-well plates, and positive wells were identified by ELISA. Specifically, the antigen was coated onto the 96-well plates at a dose of 1 μg / well. Then, the plates were blocked with 2% BSA, followed by adding 70 μL of cell serum from each well to the coated 96-well plates for incubation. Next, HRP-labeled goat anti-mouse IgG secondary antibody was added to the wells, and the plates were incubated at 37°C for 1 hour. Finally, TMB substrate solution was added to the wells for reaction, and the reaction was terminated by adding stop solution. The OD450 value was read on a microplate reader. Wells with an OD450 value greater than 3 times that of the negative control were considered positive wells.

[0251] ELISA identification results (partial):

[0252] (7) Monoclonalization

[0253] Hybridoma cells expressing high-affinity antibodies were screened and subjected to two rounds of monoclonalization. Each monoclonalization was identified by ELISA, resulting in a single-clonal hybridoma cell line. Specifically, the hybridoma cells were diluted and plated into 96-well plates using a limiting dilution method (RPMI 1640 medium pre-added with 20% serum), and ELISA was performed after 5–7 days. A total of two rounds of monoclonalization were performed.

[0254] (8) Antibody purification and affinity determination

[0255] The monoclonal hybridoma cells were expanded into T75 flasks and cultured for 5 days. The supernatant was collected, centrifuged, and filtered to remove cell debris. The antibody was then purified using a protein A affinity chromatography column. After purification, the candidate antibody was diluted to 1 ng / mL and subjected to ELISA to determine its affinity.

[0256] Partial results of ELISA affinity testing after monoclonalization:

[0257] The results show that the antibody with clone number 17G10 has a relatively high affinity, so the next step is hybridoma sequencing.

[0258] (9) Obtaining antibody sequences

[0259] The variable region sequence of the antibody was obtained using hybridoma sequencing. Specifically, total RNA was first extracted from hybridoma cells using a kit, and then cDNA was synthesized by reverse transcription using a reverse transcription kit. Next, PCR amplification was performed using mouse variable region amplification primers. After amplification, the product was purified by gel extraction, cloned into the pEASY vector, and then subjected to first-generation sequencing. All kits used in this process were from commercially available manufacturers.

[0260] The mouse Aβ1-42 antibody sequence obtained by sequencing is as follows:

[0261] Wild-type antibody heavy chain variable region amino acid sequence (SEQ ID NO:9):

[0262] CDR1:EYWPH(SEQ ID NO:11)

[0263] CDR2:AIQPLTGGTVYQDRLRD(SEQ ID NO:12)

[0264] CDR3:ALPEPYFY(SEQ ID NO:13)

[0265] Wild-type antibody light chain variable region amino acid sequence (SEQ ID NO:10):

[0266] CDR1:NSSQSLMYSQIHTMLR(SEQ ID NO:14)

[0267] CDR2:HASWKMI(SEQ ID NO:15)

[0268] CDR3:TQSTHVPIS(SEQ ID NO:16)

[0269] Implementation Example 2: Humanized Design of Mouse Aβ1-42 Antibody

[0270] Humanization design was performed using a CDR transplantation strategy. By comparing the germline gene database of heavy and light chain variable regions of human antibodies with the IMGT human antibody heavy and light chain variable regions, germline genes with high homology to the variable regions of the chimeric antibody were selected as templates. The Kabat numbering system was used to determine the amino acid number and CDR region of the antibody. The CDRs of the chimeric antibody were transplanted into the frame regions (FR) of the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The suitability of the template was comprehensively evaluated by calculating multiple factors, including template gene homology, frequency of occurrence in clinical and human trials, historical expression data, isoelectric point of the transplanted sequence, and high-risk PTM sites. The transplanted sequence of the high-scoring template was selected as the basis for reverse mutation design.

[0271] The humanized light chain templates for the chimeric antibodies were ultimately selected as IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates were IGHV1-46*01 and IGHJ6*01.

[0272] Based on the predicted variable region structure, the influence of non-conserved amino acids in the FR on the CDR conformation was quantitatively assessed. Key amino acids with high scores were reverted to the amino acids corresponding to the chimeric antibody to maintain or improve the original affinity. Different combinations of reverted mutations yielded multiple humanized variants. Specific reverted mutation designs are shown in Table 1.

[0273] Table 1: Humanized Antibody Reverse Mutation Design Note: Graft represents the insertion of the chimeric antibody CDR into the FR region sequence of the human template; Y54K indicates that the Y at position 54 of Graft is mutated to K, and so on. The reverted mutated amino acids are numbered in natural sequence.

[0274] The specific sequence of the variable region of the humanized antibody is as follows:

[0275] The amino acid sequence of VL1 is shown in SEQ ID NO:1:

[0276] The amino acid sequence of VL2 is shown in SEQ ID NO:2:

[0277] The amino acid sequence of VH1 is shown in SEQ ID NO:3:

[0278] The amino acid sequence of VH2 is shown in SEQ ID NO:4:

[0279] The amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO:5:

[0280] The amino acid sequence of the humanized light chain template IGKJ2*01 is shown in SEQ ID NO:6:

[0281] The amino acid sequence of the humanized heavy chain template IGHV1-46*01 is shown in SEQ ID NO:7:

[0282] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO:8:

[0283] By selecting different light and heavy chain sequences for cross-combination from the reverse mutation designs of the variable regions of the light and heavy chains of the aforementioned humanized antibodies, four humanized antibodies (RY-1, RY-2, RY-3, and RY-4) were finally obtained. The amino acid sequences of the variable regions of each antibody are as follows:

[0284] Table 2: Amino acid sequences corresponding to antibody variable regions

[0285] According to the Kabat numbering system, the sequence analysis results of the above-mentioned humanized antibodies VH and VL are shown in Table 3.

[0286] Table 3: Kabat analysis results of humanized antibody VH and VL sequences

[0287] According to the IMGT numbering system, the sequence analysis results of the above-mentioned humanized antibodies VH and VL are shown in Table 4.

[0288] Table 4: IMGT analysis results of humanized antibody VH and VL sequences

[0289] Example 3: Validation of the specificity and stability of humanized Aβ1-42 monoclonal antibody

[0290] (1) Recombinant expression

[0291] The original Aβ1-42 mouse antibody and four humanized antibody light and heavy chain sequences were synthesized into the pcDNA3.4 expression vector via gene synthesis. The constant region was selected to correspond to the human IgG constant region sequence. The EcoRI and HindIII restriction enzyme sites were inserted. The plasmid was then transiently transfected into Expi293 cells for expression. Specifically, the antibody amino acid sequence was first codon-optimized to adapt to 293 cells. The sequence was then directly synthesized into the vector via gene synthesis. After synthesis, the vector was transformed into E. coli DH5α competent cells. Positive clones were then selected for plasmid extraction. The light and heavy chain plasmids were then transiently transfected into Expi293F cells (available from Thermo Fisher Scientific) for antibody expression.

[0292] (2) Antigen coating and blocking

[0293] Commercially available Aβ1-42 and Aβ1-40 antigens were diluted to a concentration of 1.5 μg / mL with CB buffer, and 100 μL was coated onto each well of a colorless, transparent ELISA plate. The ELISA plates were then blocked overnight at 4°C with blocking buffer containing BSA. The Aβ1-42 and Aβ1-40 antigens were purchased from well-known domestic and international raw material companies.

[0294] (3) Indirect method for testing the specificity of humanized Aβ1-42 monoclonal antibody

[0295] After sealing, discard the liquid from the plate and blot away any residual liquid. Dilute the five expressed Aβ1-42 antibodies with PBS to concentrations of 1000 ng / mL, 250 ng / mL, 62.5 ng / mL, 15.625 ng / mL, 3.906 ng / mL, 0.977 ng / mL, 0.244 ng / mL, and 0 ng / mL, respectively. Add 100 μL to each antigen-coated ELISA plate and incubate at 37°C for 30 min. Discard the liquid from the plate and blot away any residual liquid. Wash each well five times with 300 μL of 1X washing buffer. Discard the liquid from the plate and blot away any residual liquid. Depending on the species, add 100 μL of 0.5 μg / mL enzyme-labeled goat anti-human secondary antibody or goat anti-mouse secondary antibody to each well. Incubate at 37°C for 30 min. Discard the liquid from the plate and blot away any remaining liquid. Using a plate washer, add 300 μL of 1X washing buffer to each well and wash 5 times. Discard the liquid from the plate and blot away any remaining liquid. Add 100 μL of colorimetric reagent to each well and react at 37°C for approximately 15 minutes. Add 50 μL of stop solution to each well. Measure the OD value within 15 minutes after adding the stop solution at a wavelength of 450 nm.

[0296] The test results are as follows:

[0297] The results show that among the four humanized antibodies, RY1 and RY2 have significantly enhanced affinity for the Aβ1-42 antigen; and none of the above antibodies show significant cross-linking with the Aβ1-42 antigen at concentrations below 1000 ng / mL.

[0298] (4) Verify the stability of humanized Aβ1-42 monoclonal antibody

[0299] Wild-type and four humanized Aβ1-42 monoclonal antibodies were divided into two groups and subjected to accelerated heat treatment at 4℃ and 37℃ for 14 days, respectively. The PDI index was detected using dynamic light scattering (DLS) instrumentation, and the results are as follows:

[0300] Wild-type and four humanized Aβ1-42 monoclonal antibodies were tested for their Tm values ​​in PBS buffer using a heat denaturation protein stability kit. The results are as follows:

[0301] The results show that the thermal stability of all four humanized antibodies has been improved, with RY2 and RY4 showing the greatest improvement.

[0302] Example 4: Application of humanized Aβ1-42 monoclonal antibody in the diagnosis of Alzheimer's disease

[0303] (1) HRP-labeled humanized Aβ1-42 monoclonal antibody

[0304] The preferred method is to label the humanized RY2 antibody with HRP enzyme. Specifically, the HRP enzyme is first activated with NaIO4, and then the activated HRP enzyme is coupled with the antibody in a certain proportion. NaBH4 reducing sugar is added to stabilize the Schiff base (NH4)2SO4, so that the desired enzyme is precipitated and thus the desired enzyme is obtained.

[0305] (2) Coat the Aβ amyloid antibody (recognizing epitopes 1-20aa) onto the ELISA plate.

[0306] Commercially available Aβ protein antibody (recognizing epitopes 1-20aa) was diluted to a concentration of 1.5 μg / mL with CB buffer, and 100 μL was coated into each well of a colorless, transparent ELISA plate. The ELISA plates were then blocked overnight at 4°C with blocking buffer containing BSA.

[0307] (3) ELISA kit sensitivity verification

[0308] An ELISA kit was prepared by combining HRP-labeled RY-2 antibody and commercially available Aβ amyloid antibody (recognizing epitopes 1-20aa). The sensitivity was verified using commercially available Aβ1-42 antigen. Specifically, the Aβ1-42 antigen was diluted to different concentrations: 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.625 pg / mL, 7.8125 pg / mL, 3.9 pg / mL, 1.95 pg / mL, and 0 pg / mL. 100 μL of each diluted Aβ1-42 antigen was added to an ELISA plate coated with Aβ amyloid antibody and incubated at 37°C for 60 min. The liquid in the plate was discarded and the residue was aspirated. 300 μL of 1X washing buffer was added to each well using a plate washer, and the plate was washed 5 times. The liquid in the plate was then discarded and the residue was aspirated.

[0309] Next, add 100 μL of HRP-labeled RY-2 antibody of a certain concentration to each well. Incubate at 37°C for 30 min. Discard the liquid in the plate and blot away any residual liquid. Add 300 μL of 1X washing buffer to each well using a plate washer, wash 5 times, discard the liquid in the plate and blot away any residual liquid. Add 100 μL of chromogenic solution to each well and incubate at 37°C for approximately 15 min. Then add 50 μL of stop solution to each well. Measure the OD value within 15 min after adding the stop solution at a detection wavelength of 450 nm.

[0310] The test results are as follows:

[0311] Linear fitting was performed based on the ELISA test results, and the results are shown in Figure 1.

[0312] The results showed that when humanized RY2 antibody was used in combination with commercially available Aβ amyloid antibody, it exhibited good linearity in enzyme-linked immunosorbent assay (ELISA) within a concentration range of 15 pg / mL to 500 pg / mL, with a correlation coefficient (R0.05). 2 The value is 0.995.

[0313] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-Aβ1-42 antibody or antibody binding fragment thereof, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (1) the heavy chain variable region comprises the following 3 CDRs determined according to Kabat rules: VH-CDR1 as set forth in SEQ ID NO: 11, VH-CDR2 as set forth in SEQ ID NO: 12, and VH-CDR3 as set forth in SEQ ID NO: 13; and / or the light chain variable region comprises the following 3 CDRs: VL-CDR1 as set forth in SEQ ID NO: 14, VL-CDR2 as set forth in SEQ ID NO: 15, and VL-CDR3 as set forth in SEQ ID NO: 16; or (2) the heavy chain variable region comprises the following 3 CDRs determined according to IMGT rules: VH-CDR1 as set forth in SEQ ID NO: 17, VH-CDR2 as set forth in SEQ ID NO: 18, and VH-CDR3 as set forth in SEQ ID NO: 19; and / or the light chain variable region comprises the following 3 CDRs: VL-CDR1 as set forth in SEQ ID NO: 20, VL-CDR2 as set forth in SEQ ID NO: 21, and VL-CDR3 as set forth in SEQ ID NO:

22.

2. The antibody or antibody-binding fragment thereof of claim 1, wherein, The heavy chain variable region (VH) is selected from the group consisting of: (H1) a heavy chain variable region as set forth in SEQ ID NO: 3; or (H2) a heavy chain variable region as set forth in SEQ ID NO: 4; or (H3) a heavy chain variable region as set forth in SEQ ID NO: 9; and / or The light chain variable region (VL) is selected from the group consisting of: (L1) a light chain variable region as set forth in SEQ ID NO: 1; or (L2) a light chain variable region as set forth in SEQ ID NO: 2; or (L3) a light chain variable region as set forth in SEQ ID NO:

10.

3. A recombinant protein comprising: (i) the antibody or antigen-binding fragment thereof of claim 1 or 2; and (ii) optionally, a tag sequence to assist expression and / or purification.

4. A chimeric antigen receptor, wherein, The antigen binding domain of the chimeric antigen receptor contains a single chain variable region sequence scFv targeting the Aβ peptide segment Aβ1-42, which comprises a heavy chain variable region and a light chain variable region as defined in claim 1 or 2.

5. A polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1 or 2, the recombinant protein of claim 3, or the chimeric antigen receptor of claim 4.

6. A vector containing the polynucleotide of claim 5.

7. A host cell containing the vector of claim 6 or having integrated into its genome an exogenous polynucleotide of claim 5.

8. A CAR-NK cell or CAR-T cell expressing the chimeric antigen receptor of claim 4.

9. An immunoconjugate containing: (a) an antibody moiety selected from the group consisting of: the antibody or antigen-binding fragment thereof of claim 1 or 2, the recombinant protein of claim 3, or a combination thereof; and and (b) a conjugated moiety coupled to the antibody moiety, the conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

10. Use of the antibody or antigen-binding fragment thereof of claim 1 or 2, the recombinant protein of claim 3, the chimeric antigen receptor of claim 4, the polynucleotide of claim 5, the vector of claim 6, the host cell of claim 7, the CAR-NK cell or CAR-T cell of claim 8, or the immunoconjugate of claim 9, comprising the preparation of a medicament or formulation, wherein, The drug or formulation is used for: (a) treating AD; and / or (b) clearing Aβ plaques in the brain; (c) detecting the presence or absence of Aβ in a sample.

11. A kit for detecting an Aβ1-42 peptide, comprising the antibody or antigen-binding fragment thereof of claim 1 or 2 as a first detection reagent.

12. A method for preparing a humanized anti-Aβ1-42 antibody, comprising the steps of: (a) coupling a peptide of Aβ amyloid protein at positions 37-42 to a carrier protein to obtain a conjugated carrier protein, and immunizing a mouse with the conjugated carrier protein as an antigen to obtain a mouse antibody against Aβ1-42; and (b) humanizing the non-CDR regions of the mouse antibody against Aβ1-42 to obtain a humanized anti-Aβ1-42 antibody. ​

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