Anti-p-tau217 protein monoclonal antibody, and preparation method therefor and use thereof

Abstract

Provided are a monoclonal antibody against a p-Tau217 protein and the use thereof. Specifically, provided is an antibody targeting a p-Tau217 protein or an antigen-binding fragment thereof. The provided antibody can specifically recognize and bind to a p-Tau217 protein without recognizing p-Tau181 and non-phosphorylated Tau proteins, and can be used in the preparation of a detection preparation or kit for diagnosing diseases associated with the p-Tau217 protein, such as Alzheimer's disease.

Description

A monoclonal antibody against p-Tau217 protein, its preparation method and application Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a monoclonal antibody against p-Tau217 protein, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by a gradual decline in cognitive function. In the brains of AD patients, tau protein is hyperphosphorylated, forming neurofibrillary tangles (NFTs). Phosphorylated tau protein at site 217 (p-Tau217) is considered a highly sensitive and specific biomarker for early AD. Therefore, developing antibodies that specifically recognize p-Tau217 is of great significance for the early diagnosis of AD.

[0003] Current technologies primarily employ immunological methods, using antibodies targeting specific phosphorylation sites to detect pathological changes in Alzheimer's disease (AD). For example, some studies have developed antibodies that recognize p-Tau181 and used them for AD diagnosis. However, these antibodies may have limitations in specificity and sensitivity because they may also cross-react with non-phosphorylated tau proteins or other phosphorylation sites.

[0004] While existing technologies have played a significant role in the diagnosis of Alzheimer's disease (AD), several challenges remain. First, current antibodies may lack specificity and sensitivity, potentially leading to false positives and false negatives, thus affecting diagnostic accuracy. Second, existing antibody screening methods cannot effectively exclude antibodies that cross-react with non-phosphorylated tau protein or other phosphorylation sites, which may also affect antibody specificity.

[0005] Therefore, there is an urgent need in this field for an antibody that specifically binds to the p-Tau217 protein. Summary of the Invention

[0006] The purpose of this application is to provide an antibody that specifically binds to the p-Tau217 protein, its preparation method, and its application.

[0007] Another objective of this application is to provide antibodies, chimeric antigen receptors, fusion proteins, recombinant proteins and their encoded nucleic acids, expression vectors, host cells, immunoconjugates, etc., based on p-Tau217 protein antibodies, and to provide pharmaceutical compositions including the above active ingredients.

[0008] Another objective of this application is to provide methods for preventing and / or treating diseases with high p-Tau217 protein expression, as well as diagnostic methods for diseases with high p-Tau217 protein expression.

[0009] In a first aspect, this application provides an antibody or antibody-binding fragment thereof targeting the p-Tau217 protein, 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:

[0010] (z1) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:1, VH-CDR2 shown in SEQ ID NO:2, and VH-CDR3 shown in SEQ ID NO:3; and

[0011] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:4, VL-CDR2 shown in SEQ ID NO:5, and VL-CDR3 shown in SEQ ID NO:6; or

[0012] (z2) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:7, VH-CDR2 shown in SEQ ID NO:8, and VH-CDR3 shown in SEQ ID NO:9; and

[0013] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:10, VL-CDR2 shown in SEQ ID NO:11, and VL-CDR3 shown in SEQ ID NO:12; or

[0014] (z3) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:13, VH-CDR2 shown in SEQ ID NO:14, and VH-CDR3 shown in SEQ ID NO:15; and

[0015] The light chain variable region includes the following three CDRs: VL-CDR1 shown in SEQ ID NO:16, VL-CDR2 shown in SEQ ID NO:17, and VL-CDR3 shown in SEQ ID NO:18.

[0016] 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:27; and / or

[0017] 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:28.

[0018] 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:29; and / or

[0019] 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 that of SEQ ID NO:30.

[0020] 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:31; and / or

[0021] 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:32.

[0022] 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 heavy chain framework region for connecting the heavy chain CDRs; and the light chain variable region includes the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.

[0023] In another preferred embodiment, the heavy chain variable region and the light chain variable region further include the FR region.

[0024] In another preferred embodiment, the heavy chain variable region comprises a rat- or human-derived FR region, and / or the light chain variable region comprises a rat- or human-derived FR region.

[0025] In another preferred embodiment, the heavy chain FR region includes FR-H1 shown in SEQ ID NO:19, FR-H2 shown in SEQ ID NO:20, FR-H3 shown in SEQ ID NO:21, and FR-H4 shown in SEQ ID NO:22.

[0026] In another preferred embodiment, the light chain FR region comprises FR-L1 shown in SEQ ID NO:23, FR-L2 shown in SEQ ID NO:24, FR-L3 shown in SEQ ID NO:25, and FR-L4 shown in SEQ ID NO:26. In another preferred embodiment, the antibody targeting p-Tau217 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), dual scFv, (scFv)2, microantibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein (dsFv), or combinations thereof.

[0027] In another preferred embodiment, the antibody specifically binds to the p-Tau217 protein or a derivative thereof.

[0028] In another preferred embodiment, the antibody is specifically capable of binding to p-Tau217 proteins derived from rabbits, humans, mice, and cynomolgus monkeys.

[0029] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0030] In another preferred embodiment, the light chain constant region is of human, mouse, or rabbit origin, preferably of human or rabbit origin.

[0031] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0032] In another preferred embodiment, the heavy chain constant region is of human, mouse, or rabbit origin, preferably of human or rabbit origin.

[0033] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0034] In another preferred embodiment, the antibody is a monoclonal antibody.

[0035] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies.

[0036] Secondly, this application provides a recombinant protein, wherein the recombinant protein has the following characteristics:

[0037] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of this application;

[0038] (ii) optional tag sequences to assist in expression and / or purification.

[0039] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS (SEQ ID NO:36) sequence, and a FLAG tag.

[0040] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0041] In another preferred embodiment, the recombinant protein is a fusion protein.

[0042] In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against p-Tau217 protein), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0043] In another preferred embodiment, the bispecific or multispecific antibody not only targets the p-Tau217 protein but also specifically binds to additional target antigens (such as other Alzheimer's disease antigens).

[0044] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0045] In another preferred embodiment, the recombinant protein further includes an additional fusion element (or fusion polypeptide fragment) fused together with said element (i).

[0046] Thirdly, this application provides a chimeric antigen receptor (CAR), wherein the antigen-binding domain of the chimeric antigen receptor contains an antibody single-chain variable region sequence scFv targeting the p-Tau217 protein, and the heavy chain variable region and light chain variable region of the scFv include the following complementarity-determining regions (CDRs):

[0047] (z1) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:1, VH-CDR2 shown in SEQ ID NO:2, and VH-CDR3 shown in SEQ ID NO:3; and

[0048] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:4, VL-CDR2 shown in SEQ ID NO:5, and VL-CDR3 shown in SEQ ID NO:6; or

[0049] (z2) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:7, VH-CDR2 shown in SEQ ID NO:8, and VH-CDR3 shown in SEQ ID NO:9; and

[0050] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:10, VL-CDR2 shown in SEQ ID NO:11, and VL-CDR3 shown in SEQ ID NO:12; or

[0051] (z3) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:13, VH-CDR2 shown in SEQ ID NO:14, and VH-CDR3 shown in SEQ ID NO:15; and

[0052] The light chain variable region includes the following three CDRs: VL-CDR1 shown in SEQ ID NO:16, VL-CDR2 shown in SEQ ID NO:17, and VL-CDR3 shown in SEQ ID NO:18.

[0053] Fourthly, 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 (CAR) as described in the third aspect of this application.

[0054] In another preferred embodiment, the polynucleotide comprises coding sequences for a heavy chain variable region and a light chain variable region encoding the antibody or an antigen-binding fragment thereof.

[0055] In another preferred embodiment, the polynucleotide comprises a heavy chain variable region coding sequence as shown in SEQ ID NO:5.

[0056] In another preferred embodiment, the polynucleotide comprises a light chain variable region coding sequence as shown in SEQ ID NO:13.

[0057] Fifthly, this application provides a vector containing the polynucleotide described in the fourth aspect of this application.

[0058] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0059] In another preferred embodiment, the vector is a retroviral vector.

[0060] Sixthly, this application provides a host cell containing the vector described in the fifth aspect of this application or the genome of which is integrated with exogenous polynucleotides described in the fourth aspect of this application.

[0061] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0062] In another preferred embodiment, the cell is a mammalian cell.

[0063] In another preferred embodiment, the cells are NK cells or T cells.

[0064] In another preferred embodiment, the host cell is an engineered immune cell.

[0065] In a seventh aspect, this application provides a method for preparing CAR-NK cells or CAR-T cells, wherein the CAR-NK cells or CAR-T cells express the chimeric antigen receptor described in the third aspect of this application, comprising the following steps:

[0066] 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.

[0067] Eighthly, 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.

[0068] In another preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

[0069] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0070] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 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 to 99.99% of a pharmaceutical carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0071] In another preferred embodiment, the pharmaceutical composition is used to detect Alzheimer's disease.

[0072] Ninthly, this application provides an immunoconjugate comprising:

[0073] (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

[0074] (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.

[0075] 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.

[0076] In a tenth aspect, 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 pharmaceutical composition as described in the eighth aspect of this application, or an immunoconjugate as described in the ninth aspect of this application.

[0077] (a) Preparation of detection reagents or kits; and / or

[0078] (b) To prepare drugs or formulations for the prevention and / or treatment of diseases related to p-Tau217 protein.

[0079] In another preferred embodiment, the p-Tau217 protein-related disease is Alzheimer's disease.

[0080] In another preferred embodiment, the detection reagent or kit is used to detect p-Tau217, thereby diagnosing Alzheimer's disease.

[0081] In one aspect, this application provides a method for in vitro detection of p-Tau217 protein in samples (including diagnostic or non-diagnostic samples), the method comprising the steps of:

[0082] (1) Contact the sample with the antibody or its antigen-binding fragment described in the first aspect of this application, or the recombinant protein described in the second aspect of this application;

[0083] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of p-Tau217 protein in the sample.

[0084] In another preferred embodiment, the detection is for in vitro, non-therapeutic, and non-diagnostic purposes.

[0085] In another preferred embodiment, the method is an immunochemical staning (ICC) detection method, an immunohistochemistry (IHC) detection method, a whole cell ELISA detection method, or a cell lysate ELISA detection method.

[0086] In another preferred embodiment, the method is used to diagnose Alzheimer's disease.

[0087] In a twelfth aspect, this application provides a method for preparing a recombinant polypeptide, the method comprising:

[0088] (a) Culturing the host cells described in the sixth aspect of this application under suitable expression conditions;

[0089] (b) Isolating a recombinant polypeptide from a culture, wherein the recombinant polypeptide is an antibody or its antigen-binding fragment as described in the first aspect of this application, or a recombinant protein as described in the second aspect of this application.

[0090] In a thirteenth aspect, this application provides a detection plate comprising: a substrate (support plate) and a test strip, wherein the test strip contains 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, an immunoconjugate as described in the ninth aspect of this application, or a combination thereof.

[0091] In a fourteenth aspect, this application provides a kit comprising:

[0092] (1) A first container containing the antibody or antigen-binding fragment thereof described in the first aspect of this application; and / or

[0093] (2) A second container containing a secondary antibody against the antibody;

[0094] Alternatively, the kit may contain the detection plate described in aspect thirteen of this application.

[0095] In a fifteenth aspect, this application provides a method for treating a disease associated with abnormal expression or function of the p-Tau217 protein, comprising administering to a subject in need of treatment an appropriate amount of the antibody or antigen-binding fragment thereof described in the first aspect of this application, the recombinant protein described in the second aspect of this application, the host cell described in the sixth aspect of this application, or the pharmaceutical composition described in the eighth aspect of this application.

[0096] In another preferred embodiment, the disease associated with abnormal expression or function of the p-Tau217 protein is Alzheimer's disease.

[0097] The sixteenth aspect of this application provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of this application, or the recombinant protein described in the second aspect of this application, or the host cell described in the sixth aspect of this application, and / or the pharmaceutical composition described in the eighth aspect of this application, in the preparation of a medicament for treating diseases related to abnormal expression or function of p-Tau217 protein.

[0098] In another preferred embodiment, the abnormal expression of p-Tau217 protein refers to the overexpression of p-Tau217 protein.

[0099] In another preferred embodiment, the overexpression refers to the ratio of the expression level (F1) of p-Tau217 protein to the expression level (F0) under physiological conditions (i.e., F1 / F0) being ≥1.2, preferably ≥1.5, and more preferably ≥2.00.

[0100] It should be understood that, within the scope of this application, the above-described technical features of this application 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

[0101] Figure 1 shows the agarose gel electrophoresis diagram of the PCR product of the variable region gene of the antibody in this application.

[0102] Figure 2 shows the SDS-PAGE electrophoresis diagrams after antibody purification. NR is the non-reducing electrophoresis diagram, and R is the reducing electrophoresis diagram.

[0103] Figure 3 shows the linear fitting results of the ELISA test results. Detailed Implementation

[0104] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a class of antibodies targeting the p-Tau217 protein. The antibody of this application can specifically recognize and bind to the p-Tau217 protein, and does not recognize phosphorylated proteins at other sites of the Tau protein, such as p-Tau181, nor does it recognize non-phosphorylated Tau217 protein. This application was completed based on this discovery.

[0105] Specifically, this application provides an antibody that binds to the p-Tau217 protein and its application. The antibody or its antigen-binding fragment described in this application has one or more of the following properties: 1) it binds to the p-Tau217 protein with high affinity and specificity; 2) it specifically recognizes p-Tau217 protein expressed on the cell surface; 3) the antibody of this application can be used to detect p-Tau217 protein. This application also provides a method for preparing the antibody and its application.

[0106] On one hand, this application provides multiple antibodies or their antigen-binding fragments, which include a variable region VL of the antibody light chain and a variable region VH of the antibody heavy chain, wherein the VL includes any one of VLCDR1, VLCDR2 and VLCDR3, and the VH includes any one of VHCDR1, VHCDR2 and VHCDR3, wherein the VL includes the amino acid sequence described in SEQ ID NO: 9, and the VH includes the amino acid sequence described in SEQ ID NO: 1.

[0107] In some embodiments, the antibody or its antigen-binding fragment possesses one or more properties selected from the group consisting of:

[0108] 1) It can specifically recognize the p-Tau217 protein expressed on the cell surface;

[0109] 2) Human-mouse chimeric antibodies can bind to human, monkey, and rabbit p-Tau217 proteins.

[0110] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and immunoconjugates (ADC molecules).

[0111] the term

[0112] 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. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of this application will be limited only by the appended claims.

[0113] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0114] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0115] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0116] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0117] The three-letter and single-letter codes for amino acids used in this application are as described in J. biol. chem, 243, p3558 (1968).

[0118] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0119] The term "sequence identity" as used in this application refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequence described in this application and sequences with which it has identity can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0120] 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.

[0121] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0122] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[0123] 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.

[0124] 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.

[0125] As used herein, antigen-binding fragments or antigen-binding domains will generally contain at least one variable domain. Variable domains 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. L V of domain association H In the antigen-binding fragment of the 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.

[0126] 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 loop; 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.

[0127] 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.

[0128] 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.

[0129] 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 various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire 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 backbone regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally β-sheeted and linked by three CDRs forming a linking loop, and in some cases, partially β-sheeted 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.

[0130] As those skilled in the art will know, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibody or fragment thereof of this application. This application also includes cell surface markers or antigens bound to the nanobody or fragment thereof targeting P-TAU217.

[0131] As used in this article, the terms “hypervariant region”, “highly variable region”, “complementarity determining region” and “complementarity determining region (CDR)” are used interchangeably.

[0132] 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.

[0133] 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.

[0134] In this application, the terms "nanobody of this application," "antibody of this application," "protein of this application," or "peptide of this application" are used interchangeably and all refer to peptides that specifically bind to the p-Tau217 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0135] 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.

[0136] 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 antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0137] The heavy chain variable regions of nanobodies or antibodies are of particular interest in this application because at least a portion of them are involved in binding antigens. Therefore, this application includes molecules that have antibody heavy chain variable regions with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0138] 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.

[0139] 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.

[0140] The term "antibody" in this application refers to a polypeptide containing the aforementioned CDR region that has p-Tau217 protein-binding activity. 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] Table A

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] p-Tau217 protein

[0161] p-Tau217 protein, which is Tau protein phosphorylated at the 217th threonine position, is an important biomarker for neurodegenerative diseases, showing great potential, especially in the diagnosis of Alzheimer's disease (AD).

[0162] Currently, the detection of p-Tau217 protein is mainly performed through blood tests. Compared with traditional methods based on cerebrospinal fluid (CSF) or positron emission tomography (PET), blood tests have significant advantages such as being less invasive, lower in cost, and easier to perform.

[0163] The p-Tau217 protein has demonstrated extremely high accuracy in identifying Alzheimer's disease patients. It can not only differentiate Alzheimer's disease from other neurodegenerative diseases but also predict pathological states of Alzheimer's disease, such as AβPET status and tauPET status. Furthermore, p-Tau217 protein can be used as a supplement to cerebrospinal fluid and PET scans for participant selection in clinical trials for novel disease-modifying treatments.

[0164] Anti-p-Tau217 protein antibody

[0165] In this application, the terms "antibody of this application" and "p-Tau217 protein antibody" are used interchangeably, both referring to antibodies containing heavy chain variable regions and light chain variable regions selected from the group consisting of:

[0166] (z1) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:1, VH-CDR2 shown in SEQ ID NO:2, and VH-CDR3 shown in SEQ ID NO:3; and

[0167] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:4, VL-CDR2 shown in SEQ ID NO:5, and VL-CDR3 shown in SEQ ID NO:6; or

[0168] (z2) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:7, VH-CDR2 shown in SEQ ID NO:8, and VH-CDR3 shown in SEQ ID NO:9; and

[0169] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:10, VL-CDR2 shown in SEQ ID NO:11, and VL-CDR3 shown in SEQ ID NO:12; or

[0170] (z3) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:13, VH-CDR2 shown in SEQ ID NO:14, and VH-CDR3 shown in SEQ ID NO:15; and

[0171] The light chain variable region includes the following three CDRs: VL-CDR1 shown in SEQ ID NO:16, VL-CDR2 shown in SEQ ID NO:17, and VL-CDR3 shown in SEQ ID NO:18.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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 to 20 amino acids.

[0177] The animal is preferably a mammal, such as a mouse.

[0178] The antibody in this application may be a chimeric antibody, a humanized antibody, or an antibody grafted with and / or modified with CDR that targets the p-Tau217 protein (e.g., human p-Tau217 protein, mouse p-Tau217 protein, cynomolgus monkey p-Tau217 protein, or rabbit p-Tau217 protein).

[0179] 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%.

[0180] 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.

[0181] Recombinant protein

[0182] 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.

[0183] 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.

[0184] In a preferred embodiment, the recombinant protein includes the antibody of this application.

[0185] 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.

[0186] 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 to 20 amino acids.

[0187] 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').

[0188] 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.

[0189] The single-region antibody described is a conventional single-region antibody in the art, which includes only the heavy chain variable region.

[0190] 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.

[0191] Polynucleotides

[0192] 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.

[0193] 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.

[0194] 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.

[0195] carrier

[0196] This application also provides a recombinant expression vector containing the said nucleic acid.

[0197] 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.

[0198] This application also provides a recombinant expression transformant comprising the above-mentioned recombinant expression vector.

[0199] 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.

[0200] Antibody preparation

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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).

[0209] 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.

[0210] Antibody-drug conjugates (ADCs)

[0211] As used in this article, the terms “antibody-drug conjugate”, “ADC”, and “antibody-drug conjugate” are used interchangeably.

[0212] This application also provides antibody-drug conjugates (ADCs) based on the antibodies of this application.

[0213] 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.

[0214] 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).

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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).

[0223] 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.

[0224] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0225] in:

[0226] Ab is an antibody.

[0227] LU stands for connector;

[0228] D is a drug;

[0229] Furthermore, the subscript p is a value selected from 1 to 8.

[0230] application

[0231] 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.

[0232] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal expression or function of p-Tau217 protein.

[0233] Preferably, the diagnostic agent is used to diagnose diseases associated with p-Tau217 protein expression, such as Alzheimer's disease.

[0234] In this application, the diseases associated with abnormal p-Tau217 protein expression or function are those conventionally associated with abnormal p-Tau217 protein expression or function in the art. Preferably, the diseases associated with abnormal p-Tau217 protein expression or function are Alzheimer's diseases.

[0235] Detection uses and kits

[0236] The antibody described in this application can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0237] In this application, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this application should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this application may include, for example, resected samples of tumors, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0238] The samples used in this application include fixed or preserved cell or tissue samples.

[0239] This application also provides a kit containing the antibody (or fragment thereof) of this application. In a preferred embodiment of this application, the kit further includes a container, instructions for use, buffer, etc. In a preferred embodiment, the antibody of this application can be immobilized on a detection plate.

[0240] The kit described in this application is used to detect p-Tau217 protein, thereby diagnosing Alzheimer's disease.

[0241] Pharmaceutical Composition

[0242] 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.

[0243] 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.

[0244] 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).

[0245] The pharmaceutical composition described in this application is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal expression or function of p-Tau217 protein.

[0246] The pharmaceutical composition of this application can be directly used to bind to the p-Tau217 protein molecule, and therefore can be used for the prevention and treatment of diseases such as tumors.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] This application provides the use of the above-described pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal expression or function of p-Tau217 protein. Preferably, the disease associated with abnormal expression or function of p-Tau217 protein is Alzheimer's disease.

[0252] The main advantages of this application include:

[0253] 1. The antibody of this application has high specificity and can specifically bind to the p-Tau217 protein. Developing a p-Tau217 protein detection method based on the antibody of this application can effectively avoid false positive results and improve the accuracy of the detection method.

[0254] 2. The p-Tau217 protein detection method developed using the antibody of this application has high sensitivity and good linearity. It can detect low concentrations of p-Tau217 protein, which is beneficial for the early diagnosis of Alzheimer's disease.

[0255] 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. Percentages and parts are by weight unless otherwise stated.

[0256] Example 1: Preparation of rabbit monoclonal antibody against p-Tau217 protein

[0257] Step 1: Antigen Preparation

[0258] The p-Tau 217 polypeptide region was selected from positions 208-227 of the Tau protein, with the following sequence:

[0259] SRSRTPSLP{pT}PPTREPKKVA (SEQ ID NO:33), in which threonine is phosphorylated at position 217;

[0260] The p-Tau 181 polypeptide region was selected from positions 173-191 of the Tau protein, with the following sequence:

[0261] AKTPPAPK{pT}PPSSGEPPKS (SEQ ID NO:34), in which threonine is phosphorylated at position 181;

[0262] The Tau protein region from position 208 to 227 was selected as the non-phosphorylated 217 polypeptide region, with the following sequence:

[0263] SRSRTPSLPTPPTREPKKVA (SEQ ID NO:35) is synthesized by adding the amino acid GGC at the C-terminus of the p-Tau 217 polypeptide to link the carrier protein KLH as an immunogen.

[0264] During the synthesis of the p-Tau 217 polypeptide, the amino acid GGC was added to the C-terminus to link biotin as a positive screening antigen.

[0265] When synthesizing the non-phosphorylated Tau 217 polypeptide, the amino acid GGC was added to the C-terminus to link biotin as a negative screening antigen.

[0266] During the synthesis of the p-Tau 181 polypeptide, the amino acid GGC was added to the C-terminus to link biotin as a negative screening antigen.

[0267] Step 2: Antigen Immunization

[0268] Healthy New Zealand white rabbits were selected, and the antigen dosage was calculated at 500 μg per rabbit per immunization. For the initial immunization, the immunogen was mixed with an equal volume of Freund's complete adjuvant to prepare an emulsion, which was injected subcutaneously at multiple sites. Two weeks later, 500 μg of the immunogen was mixed with an equal volume of Freund's unadjuvant to prepare an emulsion, which was also injected subcutaneously at multiple sites for two booster immunizations. After the three immunizations, peripheral blood samples were collected from the rabbits, coated with p-Tau 217 peptide unconjugated with KLH, and serum titers were determined by ELISA. Rabbits with high serum titers were selected, and a booster immunization was performed by subcutaneous injection of 500 μg of immunogen at multiple sites. 15 mL of fresh rabbit peripheral blood was then collected in an anticoagulant tube.

[0269] Step 3: Isolation of rabbit peripheral blood PBMCs

[0270] Dilute whole blood with an equal volume of whole blood and tissue diluent, and aliquot into two 50 mL centrifuge tubes. Add 20 mL of separation buffer to each tube, and spread the diluted blood evenly on top of the separation buffer, ensuring a clear interface between the two liquids. Centrifuge at room temperature, 800 × g, for 30 min. After centrifugation, distinct layers will appear: the top layer is diluted plasma, the middle layer is clear separation buffer, the white membrane layer between the plasma and separation buffer is the lymphocyte layer, and the bottom of the centrifuge tube contains red blood cells and granulocytes. Carefully aspirate the white membrane cells into a clean 15 mL centrifuge tube and wash the cells with 10 mL of PBS. Centrifuge at 250 g for 10 min. Discard the supernatant, resuspend the cells in 5 mL of PBS or cell washing buffer, and centrifuge at 250 × g for 10 min. Repeat the washing process once more, discard the supernatant, and resuspend the cells in 1 mL of MACS buffer.

[0271] Step 4: Enrichment and Activation of Rabbit Memory B Cells

[0272] Rabbit memory B cells expressing IgG were sorted according to the rabbit memory B cell sorting procedure. Cells were then collected by centrifugation, resuspended in rabbit memory B cell activation medium, and seeded into 24-well plates. The plates were incubated at 37°C for 5 days in a 5% CO2 incubator, and fresh rabbit memory B cell activation medium was added after 48 hours.

[0273] Step 5: Screening of rabbit mono-B cells

[0274] Following the single B cell screening procedure, activated plasma B cells are introduced into the instrument, and then positive and negative antigens are incubated with the activated rabbit plasma B cells. Rabbit plasma B cells that can secrete non-cross-reactive antibodies are screened out by the fluorescence signal of the instrument.

[0275] Step Six: Export rabbit single B cells to complete antibody variable region sequence amplification.

[0276] Selected rabbit single B cells were introduced into 96-well plates using software, one cell per well. Single-cell cDNA amplification was then performed using a single-cell cDNA amplification kit to obtain the single B cell cDNA sequence. The antibody light and heavy chain variable regions were then amplified by PCR and subsequently sequenced.

[0277] Figure 1 shows the agarose gel electrophoresis bands of the antibody variable region gene PCR products. The boxes in the figure represent the successfully sequenced antibody pairs. The sequencing results are shown in Table 1.

[0278] Table 1

[0279] Step 7: Recombinant Expression of Antibodies

[0280] The antibody's light and heavy chain sequences were synthesized into the pcDNA3.4 expression vector via gene synthesis, with EcoRI and HindIII restriction enzyme sites inserted. The plasmid was then transiently transfected into Expi293 cells for expression. Specifically, the antibody amino acid sequence was first codon-optimized to fit Expi293 cells. The sequence was then synthesized into a vector, which was subsequently transformed into *E. coli* DH5α competent cells. Positive clones were then selected for plasmid extraction using the Tiangen Plasmid Mini-Prep Kit. The light and heavy chain plasmids were then transiently transfected into Expi293F cells for antibody expression.

[0281] Step 8: Antibody Purification

[0282] Cell supernatant was collected 5–7 days after transfection and purified to obtain recombinant antibodies. Specifically, before purification, the cell supernatant was centrifuged at 4000 rpm for 20 minutes, then filtered through a 0.22 μm polypropylene membrane, and finally purified using an AKTA Pure purifier and a Protein A affinity purification column.

[0283] After purification, the purity and integrity of the antibody samples were detected and verified by SDS-PAGE electrophoresis combined with staining analysis.

[0284] Figure 2 shows the SDS-PAGE results after antibody purification. NR represents non-reducing electrophoretic staining, and R represents reducing electrophoretic staining.

[0285] Example 2: Specificity Validation of p-Tau217 Monoclonal Antibody

[0286] 2.1 Method

[0287] Step 1: Antigen coating and blocking

[0288] Commercially available p-Tau217 full-length antigen (recombinant antigen), p-Tau181 full-length antigen (recombinant antigen), and non-phosphorylated total-Tau antigen (recombinant antigen) 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.

[0289] Step 2: Indirect method for testing the specificity of p-Tau217 monoclonal antibody

[0290] After sealing, discard the liquid from the plate and blot away any residual liquid. Dilute the p-Tau217 monoclonal antibody with PBS in the following dilution gradients: 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. Add 100 μL to each of the three antigen-coated ELISA plates and incubate at 37°C for 30 min. Discard the liquid from the plate and blot away any residual liquid. Wash each well with 300 μL of 1X washing buffer five times. Discard the liquid from the plate and blot away any residual liquid. Add 100 μL of 0.5 μg / mL enzyme-labeled goat anti-rabbit 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.

[0291] 2.2 Results

[0292] Table 1

[0293] Table 2

[0294] Table 3

[0295] As shown in Table 1-3, the three screened p-Tau217 monoclonal antibodies have very strong specificity to the commercially available p-Tau217 antigen and no cross-reactivity with the non-phosphorylated total-Tau217 antigen and p-Tau181.

[0296] Example 3 Affinity Test

[0297] Step 1: CM5 Chip Antigen Pre-enrichment: Dilute the full-length p-Tau217 antigen (recombinant antigen) to 20 μg / mL with sodium acetate solutions at pH 5.0, pH 4.5, and pH 4.0 respectively, then inject them into the chip and detect changes in the instrument's RU value. Based on the results, dilute the antigen to 10 μg / mL with sodium acetate solution at pH 4.5 for the next step of chip-conjugated antigen.

[0298] Step 2: Antigen conjugation: The commercially available p-Tau217 recombinant antigen (Absea, PC006) is conjugated onto the chip. First, the antigen is diluted to 10ug / mL with sodium acetate solution at pH 4.5. Take 200ul and conjugate it onto the chip. Wait 30min, and the instrument will automatically complete the conjugation of the antigen and the chip.

[0299] Step 3: Multi-cycle kinetic analysis: The three screened p-Tau217 rabbit monoclonal antibodies were diluted with HBS-EP+ buffer to concentration gradients of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, and 0 ng / mL. The concentrations were then sequentially analyzed using an SPR assay instrument (Cytiva, Biacore T200). The antibody affinity constant KD was ultimately determined by analyzing the antigen-antibody binding constant Ka and the dissociation constant Kd.

[0300] The measurement results are shown in Table 4.

[0301] Table 4

[0302] As shown in Table 4, the affinity of the p-Tau217 rabbit monoclonal antibodies screened in this application all reached a high level.

[0303] Example 4: Application of p-Tau217 monoclonal antibody in the diagnosis of Alzheimer's disease

[0304] Step 1: HRP-labeled p-Tau217 monoclonal antibody

[0305] Antibody 1 was labeled with HRP enzyme. The specific method was to first activate HRP enzyme with NaIO4, then couple the activated HRP enzyme with the antibody in a certain proportion, add NaBH4 reducing sugar to stabilize Schiff base (NH4)2SO4, and precipitate the desired enzyme to obtain the desired enzyme.

[0306] Step 2: Coat the Tau protein antibody onto the ELISA plate.

[0307] Commercially available total-Tau protein antibody was 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.

[0308] The HRP-labeled p-Tau217 monoclonal antibody and the commercially available total-Tau protein antibody were combined to create an ELISA kit.

[0309] Step 3: ELISA kit sensitivity verification

[0310] Cerebrospinal fluid samples were collected from clinical AD patients. The collected cerebrospinal fluid samples were sent to the Simoa platform for value determination. After the value determination was completed, the clinical samples were serially diluted using sample diluents. The dilution concentrations are shown in Table 5.

[0311] Table 5

[0312] Add 100 μL of clinical samples of different concentrations to ELISA plates coated with total-Tau protein antibody and incubate at 37°C for 60 min. Discard the liquid from the plate and blot away any remaining liquid. Add 300 μL of 1X washing buffer to each well using a plate washer and wash 5 times. Discard the liquid from the plate and blot away any remaining liquid.

[0313] Next, add 100 μL of HRP-labeled antibody at a certain concentration to each well and incubate at 37°C for 30 min. Discard the liquid from the plate and blot away any remaining residue. 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 residue. Add 100 μL of chromogenic buffer 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.

[0314] The test results are shown in Table 6.

[0315] Table 6

[0316] As shown in Table 6 and Figure 3, when p-Tau217 monoclonal antibody is used in combination with commercially available total-Tau protein antibody, it exhibits good linearity in enzyme-linked immunosorbent assay (ELISA) within a concentration range of 1.0 pg / mL to 100 pg / mL, with a correlation coefficient (R0). 2 The value is 0.9924. This means that when the p-Tau217 monoclonal antibody screened in this application is paired with commercially available high-affinity total-Tau protein antibodies, it can be used for the diagnosis of Alzheimer's disease.

[0317] In addition to its application in ELISA methodology development, this antibody can also be used on other diagnostic platforms, including chemiluminescence and fluorescence immunochromatography. Therefore, the p-Tau217 monoclonal antibody of this application has significant potential in assisting the diagnosis of Alzheimer's disease.

[0318] 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 antibody targeting p-Tau217 protein, or an antibody-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (z1) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:1, VH-CDR2 shown in SEQ ID NO:2, and VH-CDR3 shown in SEQ ID NO:3; and The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:4, VL-CDR2 shown in SEQ ID NO:5, and VL-CDR3 shown in SEQ ID NO:6; or (z2) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:7, VH-CDR2 shown in SEQ ID NO:8, and VH-CDR3 shown in SEQ ID NO:9; and The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:10, VL-CDR2 shown in SEQ ID NO:11, and VL-CDR3 shown in SEQ ID NO:12; or (z3) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:13, VH-CDR2 shown in SEQ ID NO:14, and VH-CDR3 shown in SEQ ID NO:15; and The light chain variable region includes the following three CDRs: VL-CDR1 shown in SEQ ID NO:16, VL-CDR2 shown in SEQ ID NO:17, and VL-CDR3 shown in SEQ ID NO:

18.

2. A recombinant protein comprising: (i) the antibody or antigen-binding fragment thereof as described in claim 1; (ii) optional tag sequences to assist in expression and / or purification.

3. A chimeric antigen receptor, wherein its antigen-binding domain contains an antibody single-chain variable region sequence scFv targeting the p-Tau217 protein, wherein, The heavy chain variable region and light chain variable region of the scFv include the following complementary determinant regions (CDRs): (z1) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:1, VH-CDR2 shown in SEQ ID NO:2, and VH-CDR3 shown in SEQ ID NO:3; and The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:4, VL-CDR2 shown in SEQ ID NO:5, and VL-CDR3 shown in SEQ ID NO:6; or (z2) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:7, VH-CDR2 shown in SEQ ID NO:8, and VH-CDR3 shown in SEQ ID NO:9; and The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:10, VL-CDR2 shown in SEQ ID NO:11, and VL-CDR3 shown in SEQ ID NO:12; or (z3) The heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:13, VH-CDR2 shown in SEQ ID NO:14, and VH-CDR3 shown in SEQ ID NO:15; and The light chain variable region includes the following three CDRs: VL-CDR1 shown in SEQ ID NO:16, VL-CDR2 shown in SEQ ID NO:17, and VL-CDR3 shown in SEQ ID NO:

18.

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

5. A carrier comprising the polynucleotide of claim 4.

6. A host cell containing the vector of claim 5 or the genome thereof having an exogenous polynucleotide of claim 4 integrated therein.

7. A method for preparing CAR-NK cells or CAR-T cells, wherein, The CAR-NK cells or CAR-T cells expressing the chimeric antigen receptor of claim 3 include the following steps: The polynucleotide of claim 4 or the vector of claim 5 is introduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

8. A method for the diagnostic or non-diagnostic in vitro detection of p-Tau217 protein in a sample, comprising the following steps: (1) Contact the sample with the antibody or its antigen-binding fragment as described in claim 1, or the recombinant protein as described in claim 2; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of p-Tau217 protein in the sample.

9. An immunoconjugate comprising: (a) Antibody portion, wherein the antibody portion is selected from the group consisting of: The antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 2, or a combination thereof; and (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.

10. Use of an antibody or antigen-binding fragment thereof as claimed in claim 1, a recombinant protein as claimed in claim 2, a chimeric antigen receptor as claimed in claim 3, a polynucleotide as claimed in claim 4, a vector as claimed in claim 5, a host cell as claimed in claim 6, or an immunoconjugate as claimed in claim 9, comprising: (a) Preparation of detection reagents or kits; and / or (b) To prepare drugs or formulations for the prevention and / or treatment of diseases related to p-Tau217 protein.

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