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

Abstract

The present invention provides a monoclonal antibody against the p-Tau217 protein and its applications. Specifically, the present invention provides an antibody targeting the p-Tau217 protein or an antigen-binding fragment thereof. The antibody provided by the present invention can specifically recognize and bind to the p-Tau217 protein, and does not recognize p-Tau181 and non-phosphorylated Tau protein, and can be used to prepare detection preparations or kits for diagnosing p-Tau217 protein-related diseases, such as Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a monoclonal antibody against p-Tau217 protein, its preparation method and application. Background Art

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

[0003] Existing technologies mainly adopt immunological methods to detect pathological changes in AD by preparing antibodies against specific phosphorylation sites. For example, some studies have developed antibodies that can recognize p-Tau181 and used them for the diagnosis of AD. However, these antibodies may have certain limitations in terms of specificity and sensitivity, because they may also cross-react with non-phosphorylated tau protein or other phosphorylation sites.

[0004] Although existing technologies have played an important role in the diagnosis of AD, there are still some challenges. First, existing antibodies may be insufficient in terms of specificity and sensitivity, which may lead to false positive and false negative results, thus affecting the accuracy of diagnosis. 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 the specificity of the antibodies.

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

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

[0007] Another object of the present invention is to provide antibodies, chimeric antigen receptors, fusion proteins, recombinant proteins and their encoding nucleic acids, expression vectors, host cells, immunoconjugates, etc. based on the p-Tau217 protein antibody, and to provide a pharmaceutical composition comprising the above active ingredients.

[0008] Another object of the present invention is to provide a method for preventing and / or treating diseases with high expression of p-Tau217 protein, as well as a diagnostic method for diseases with high expression of p-Tau217 protein.

[0009] In a first aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof that targets the p-Tau217 protein, wherein the antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region and a light-chain variable region selected from the following groups:

[0010] (z1) The heavy-chain variable region comprises the following 3 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 3 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 3 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 3 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 3 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 comprises the following 3 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 that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:27; and / or

[0017] The antibody or antigen-binding fragment comprises a light chain variable region having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:29; and / or

[0019] The antibody or antigen-binding fragment comprises a light chain variable region having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to 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 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:31; and / or

[0021] The antibody or antigen-binding fragment comprises a light chain variable region having an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:32.

[0022] In another preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the three heavy chain CDRs and the heavy chain framework regions for connecting the heavy chain CDRs; the light chain variable region comprises the three light chain CDRs and the light chain framework regions for connecting the light chain CDRs.

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

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

[0025] In another preferred embodiment, the heavy chain FR region comprises 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 or antigen-binding fragment thereof targeting p-Tau217 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"), bis-scFv, (scFv)2, minibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein ("dsFv"), or a combination 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 comprises a light chain constant region.

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

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

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

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

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

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

[0036] In a second aspect of the present invention, there is provided a recombinant protein having:

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

[0038] and (ii) optionally, a tag sequence for assisting expression and / or purification.

[0039] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS 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 the p-Tau217 protein), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

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

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

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

[0046] In the third aspect of the present invention, a chimeric antigen receptor CAR is provided. The antigen-binding domain of the chimeric antigen receptor contains a single-chain variable region sequence scFv of an antibody targeting the p-Tau217 protein. The heavy-chain variable region and the light-chain variable region of the scFv include the following complementarity-determining regions CDR:

[0047] (z1) The heavy-chain variable region contains the following 3 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 contains the following 3 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 contains the following 3 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 contains the following 3 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 comprises 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] In a fourth aspect of the present invention, there is provided a polynucleotide encoding the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, or the chimeric antigen receptor CAR as described in the third aspect of the present invention.

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

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

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

[0057] In a fifth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the fourth aspect of the present invention.

[0058] In another preferred embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

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

[0060] In a sixth aspect of the present invention, there is provided a host cell containing the vector as described in the fifth aspect of the present invention or having the polynucleotide as described in the fourth aspect of the present invention integrated into its genome exogenously.

[0061] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

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

[0063] In another preferred embodiment, the cell is an NK cell or a T cell.

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

[0065] In the seventh aspect of the present invention, a method for preparing CAR-NK cells or CAR-T cells is provided. The CAR-NK cells or CAR-T cells express the chimeric antigen receptor described in the third aspect of the present invention, and the method includes the following steps:

[0066] Transfecting the polynucleotide described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

[0067] In the eighth aspect of the present invention, a pharmaceutical composition is provided. The pharmaceutical composition contains the antibody or its antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the chimeric antigen receptor described in the third aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the host cell described in the sixth aspect of the present invention, or a combination thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0068] In another preferred example, the pharmaceutical composition is a preparation, preferably a liquid preparation.

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

[0070] In another preferred example, the pharmaceutical composition includes 0.01-99.99% of the antibody or its antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the chimeric antigen receptor described in the third aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the host cell described in the sixth aspect of the present invention, or a combination thereof, and 0.01-99.99% of a pharmaceutical carrier, and the percentages are by mass of the pharmaceutical composition.

[0071] In another preferred example, the pharmaceutical composition is used for detecting Alzheimer's disease.

[0072] In the ninth aspect of the present invention, an immunoconjugate is provided. The immunoconjugate contains:

[0073] (a) An antibody moiety selected from the group consisting of the antibody or its antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or a combination thereof; and

[0074] (b) A conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0075] In another preferred embodiment, the conjugate is selected from: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme, radionuclide, biotoxin, cytokine (such as IL-2, etc.), antibody, antibody Fc fragment, antibody scFv fragment, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, etc.

[0076] In a tenth aspect of the present invention, there is provided the use of the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the vector as described in the fifth aspect of the present invention, the host cell as described in the sixth aspect of the present invention, the pharmaceutical composition as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention,

[0077] (a) for preparing a detection reagent or kit; and / or

[0078] (b) for preparing a drug or preparation for preventing and / or treating p-Tau217 protein-related diseases.

[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 an eleventh aspect of the present invention, there is provided a method for in vitro detecting (including diagnostic or non-diagnostic) p-Tau217 protein in a sample, the method comprising the steps of:

[0082] (1) contacting the sample with the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention;

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

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

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

[0086] In another preferred example, the method is used for diagnosing Alzheimer's disease.

[0087] In the twelfth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0088] (a) Culturing the host cell described in the sixth aspect of the present invention under conditions suitable for expression;

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

[0090] In the thirteenth aspect of the present invention, a detection plate is provided, the detection plate comprising: a substrate (support plate) and a test strip, wherein the test strip contains the antibody or its antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the immunoconjugate described in the ninth aspect of the present invention, or a combination thereof.

[0091] In the fourteenth aspect of the present invention, a kit is provided, the kit comprising:

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

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

[0094] Alternatively, the kit contains the detection plate described in the thirteenth aspect of the present invention.

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

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

[0097] In the sixteenth aspect of the present invention, there is provided the use of the antibody or its antigen-binding fragment described in the first aspect of the present invention, or the recombinant protein described in the second aspect of the present invention, or the host cell described in the sixth aspect of the present invention and / or the pharmaceutical composition described in the eighth aspect of the present invention in the preparation of a medicament for treating diseases associated with abnormal expression or function of p-Tau217 protein.

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

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

[0100] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 The agarose gel electrophoresis pattern of the PCR product of the antibody variable region gene of the present invention is shown.

[0102] Figure 2 The SDS-PAGE electrophoresis pattern after antibody purification is shown, where NR is the non-reducing electrophoresis pattern and R is the reducing electrophoresis pattern.

[0103] Figure 3 The linear fitting result of the ELISA detection result is shown. DETAILED DESCRIPTION OF THE INVENTION

[0104] Through extensive and in-depth research, and through a large number of experiments and screenings, the present inventors unexpectedly discovered for the first time a class of antibodies targeting the p-Tau217 protein. The antibodies of the present invention can specifically recognize and bind to the p-Tau217 protein, and do not recognize proteins phosphorylated at other sites of the Tau protein, such as p-Tau181, nor do they recognize non-phosphorylated Tau217 protein. Based on this, the present invention was completed.

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

[0106] On the one hand, the present invention provides a plurality of antibodies or antigen-binding fragments thereof, which comprise an antibody light chain variable region VL and an antibody heavy chain variable region VH. The VL comprises any one of VLCDR1, VLCDR2, and VLCDR3, and the VH comprises any one of VHCDR1, VHCDR2, and VHCDR3. Wherein the VL comprises the amino acid sequence of SEQ ID NO: 9, and the VH comprises the amino acid sequence of SEQ ID NO: 1.

[0107] In certain embodiments, the antibody or antigen-binding fragment thereof has one or more properties selected from the group consisting of:

[0108] 1) capable of specifically recognizing p-Tau217 protein expressed on the cell surface;

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

[0110] In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibody, chimeric antibody, humanized antibody, and immunoconjugate (ADC molecule).

[0111] The term

[0112] To more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0113] As used herein, the term "comprising" or its variants such as "including" or "including having" etc. are understood to include the stated element or component, without excluding other elements or other components.

[0114] The term "about" may refer to a value or a component within an acceptable error range of a specific value or component determined by those of ordinary skill in the art, which will depend in part on how the value or component 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 specified, any concentration range, percentage range, ratio range or integer range shall be understood to include any integer value within the said range and, where appropriate, fractional values thereof (e.g., one tenth and one hundredth of an integer).

[0116] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the related listed items.

[0117] The three-letter and single-letter codes of amino acids used in the present invention are as described in J.biol.chem, 243, p3558 (1968).

[0118] As used herein, the term "optionally" or "optionally" means that the subsequent described event or situation may occur but is not necessarily required to occur.

[0119] "Sequence identity" as described in the present invention 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 sequences described in the present invention and the sequences having identity therewith can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0120] As used herein, the term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises 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 carboxy 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 herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0122] As used herein, the term "light chain variable region" is used interchangeably with "VL".

[0123] As used herein, terms such as "antigen-binding domain" include any naturally occurring, enzymatically obtainable, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains. Such DNA is known and / or readily obtainable 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 by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains in a suitable configuration, 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 that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0125] As used herein, an antigen-binding fragment or antigen-binding domain will generally comprise at least one variable domain. The variable domain can have any size or amino acid composition and will typically comprise at least one CDR that is contiguous with or in-frame with one or more framework sequences. In an antigen-binding fragment having a V L domain associated with a V H domain, the VH and VL domains can be arranged relative to each other in any suitable arrangement. 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 a monomeric VH or VL domain.

[0126] In the amino acid sequence of the variable region of the light chain or heavy chain of a given antibody, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, including, for example: Chothia, which is based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983)), AbM (University of Bath), Contact (University College London), the international Immuno GeneTics database (IMGT), the EU numbering system, and the Chothia definition based on the position of loop structures.

[0127] It should be understood that the precise amino acid sequence boundaries of the CDRs in the present invention can optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise stated, in the present invention, when referring to the residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbered positions according to the Kabat numbering system.

[0128] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric protein of approximately 150,000 daltons with the same structural features, which consists 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, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by a plurality of constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0129] As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions in the variable regions are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration, connected by three CDRs that form connecting loops and can form a partial β-sheet structure in some cases. The CDRs in each chain are brought closely 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, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0130] As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the nanobodies or fragments thereof against P-TAU217 described above.

[0131] As used herein, the terms "hypervariable region", "highly variable region", "complementary determining region", and "complementarity determining region (CDR)" are used interchangeably.

[0132] In a preferred embodiment of the present invention, the nanobody or the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0133] In a preferred embodiment of the present invention, the nanobody or the heavy chain of the antibody comprises the above-mentioned heavy chain variable region and a heavy chain constant region.

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

[0135] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention 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 at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present invention.

[0136] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the heavy chain variable region, called complementarity-determining regions (CDRs), which divide this segment into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures that are brought close to each other in spatial structure by the β-sheets formed by the intervening FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

[0137] The nanobodies or heavy chain variable regions of the antibodies of the present invention are of particular interest because at least some of them are involved in antigen binding. Accordingly, the present invention includes those molecules having a heavy chain variable region of an antibody with CDRs, provided that the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0138] The present invention includes not only intact antibodies, but also fragments of antibodies having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives, and analogs of the said antibodies.

[0139] As used herein, the terms "fragment", "derivative", and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative 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 a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that extends the polypeptide half-life, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0140] The antibody of the present invention refers to a polypeptide having p-Tau217 protein-binding activity and including the above CDR regions. This term also includes variant forms of the polypeptide containing the above CDR regions that have the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or close properties, the function of the protein is usually not changed. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the antibody of the present invention.

[0141] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using the antiserum against the antibody of the present invention.

[0142] The present invention also provides other polypeptides, such as fusion proteins containing the antibody or its fragments. In addition to almost full-length polypeptides, the present invention also includes fragments of the antibody of the present invention. Usually, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, 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.

[0143] In the present invention, the "conservative variant of the antibody of the present invention" refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids in the amino acid sequence of the antibody of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0144] Table A

[0145]

[0146]

[0147] The present invention also provides polynucleotide molecules encoding the above antibody or its fragments or its fusion proteins. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0148] The polynucleotides encoding the mature polypeptide of the present invention include: a coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0149] The term "polynucleotide encoding a polypeptide" may be a polynucleotide including the polynucleotide encoding this polypeptide, or may also be a polynucleotide further including additional coding and / or non-coding sequences.

[0150] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides described in the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least above 90%, preferably above 95%. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptide.

[0151] The full-length nucleotide sequence or fragments thereof of the antibody of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis methods. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Usually, very long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0152] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually to clone them into a vector, then transfer them into cells, and then separate the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0153] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0154] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0155] The host cell can be a prokaryotic cell, such as a bacterial cell; or 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; animal cells such as CHO, COS7, 293 cells, etc.

[0156] Transformation of the host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0157] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0158] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods utilizing its 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 renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0159] The nanobody or antibody of the present invention can be used alone, or can be conjugated or coupled with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety or a combination of any of the above substances.

[0160] Detectable labels for diagnostic purposes include but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0161] Therapeutic agents that can bind to or be conjugated with the antibodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0162] p-Tau217 protein

[0163] p-Tau217 protein, that is, Tau protein phosphorylated at the 217th threonine position, is an important biomarker for neurodegenerative diseases and shows great potential especially in the diagnosis of Alzheimer's disease (AD).

[0164] Currently, the detection of p-Tau217 protein is mainly carried out through blood tests. Compared with traditional methods based on cerebrospinal fluid (CSF) or positron emission tomography (PET), blood tests have significant advantages such as less invasiveness, lower cost, and ease of operation.

[0165] p-Tau217 protein shows extremely high accuracy in identifying patients with Alzheimer's disease. It can not only be used to distinguish Alzheimer's disease from other neurodegenerative diseases, but also to predict the pathological status of Alzheimer's disease, such as AβPET status, tau PET status, etc. In addition, p-Tau217 protein can also be used as a supplement to CSF and PET for participant selection in clinical trials of novel disease-modifying therapies.

[0166] Anti-p-Tau217 protein antibody

[0167] In the present invention, the terms "antibodies of the present invention" and "p-Tau217 protein antibodies" are used interchangeably and both refer to antibodies comprising a heavy chain variable region and a light chain variable region selected from the group consisting of:

[0168] (z1) The heavy chain variable region comprises the following 3 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

[0169] The light chain variable region comprises the following 3 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

[0170] (z2) The heavy chain variable region contains 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

[0171] The light chain variable region contains 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

[0172] (z3) The heavy chain variable region contains 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

[0173] The light chain variable region contains 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.

[0174] Preferably, the antibody described herein is one or more of an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody fragment, scFv), a single domain antibody (singledomain antibody, sdAb), and a single region antibody (Signle-domain antibody), as well as monoclonal antibodies or polyclonal antibodies prepared from the above antibodies. The monoclonal antibodies can be developed by a variety of methods and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

[0175] The antibody full-length protein described is a conventional antibody full-length protein in the art, which includes 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 constitute a fully human antibody full-length protein with a human heavy chain constant region and a human light chain constant region. Preferably, the antibody full-length protein is IgG1, IgG2, IgG3, or IgG4.

[0176] The antibody of the present invention can be a double-stranded or single-stranded antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0177] The antibody derivatives of the present invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the art, and any one or more of IgA, IgD, IgE, IgG and IgM antibodies or antibodies of other subtypes.

[0178] The single-chain antibody described is a conventional single-chain antibody in the art, which includes a heavy-chain variable region, a light-chain variable region and a short peptide of 15 to 20 amino acids.

[0179] Among them, the animal is preferably a mammal, such as a mouse.

[0180] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR grafted and / or modified antibody targeting the p-Tau217 protein (such as human p-Tau217 protein, mouse p-Tau217 protein, cynomolgus monkey p-Tau217 protein or rabbit p-Tau217 protein).

[0181] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0182] In the above content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0183] Recombinant protein

[0184] The present invention also provides a recombinant protein, which includes 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 the present invention, 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 the present invention.

[0185] 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, 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 light-chain antibody constant region or a human-derived antibody light-chain constant region, more preferably a human-derived antibody light-chain constant region.

[0186] In a preferred example, the recombinant protein includes the antibody of the present invention.

[0187] The recombinant protein described above is a conventional protein in the art. Preferably, it is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single-chain antibody (single chain antibody fragment, scFv), a single-domain antibody (single domain antibody, sdAb), and a single-region antibody (Single-domain antibody), as well as monoclonal antibodies or polyclonal antibodies prepared from the above antibodies.

[0188] The single-chain antibody described above is a conventional single-chain antibody in the art, which includes a heavy-chain variable region, a light-chain variable region, and a short peptide of 15-20 amino acids.

[0189] The antigen-antibody binding domain protein fragment described above is a conventional antigen-antibody binding domain protein fragment in the art, which includes a light-chain variable region, a light-chain constant region, and an Fd segment of a heavy-chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0190] The single-domain antibody described above is a conventional single-domain antibody in the art, which includes a heavy-chain variable region and a heavy-chain constant region.

[0191] The single-region antibody described above is a conventional single-region antibody in the art, which only includes a heavy-chain variable region.

[0192] Among them, the preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: isolating from an expression transformant that recombinantly expresses the protein or obtaining it by artificially synthesizing a protein sequence. The preferred method for isolating from an expression transformant that recombinantly expresses the protein is as follows: cloning a polynucleotide molecule encoding the protein and having 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.

[0193] Polynucleotide

[0194] The present invention also provides a polynucleotide that encodes the antibody or recombinant protein of the present invention described above or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

[0195] 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 the method of artificial total sequence synthesis.

[0196] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a homolog of the polynucleotide. The homologs of the polynucleotides in the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining antibody activity.

[0197] vector

[0198] The present invention also provides a recombinant expression vector comprising the said nucleic acid.

[0199] Among them, the recombinant expression vector can be obtained by conventional methods in the art, that is: constructed by ligating the nucleic acid molecule of the present invention to various expression vectors. The said expression vectors are various conventional vectors in the art, as long as they can accommodate the aforementioned nucleic acid molecule. Preferably, the said vectors include: various plasmids, cosmids, phages or viral vectors, etc.

[0200] The present invention also provides a recombinant expression transformant comprising the above-mentioned recombinant expression vector.

[0201] Among them, the preparation method of the said recombinant expression transformant is a conventional preparation method in the art, preferably: prepared by transforming the above-mentioned recombinant expression vector into a host cell. The said host cells are various conventional host cells in the art, as long as they can satisfy the stable self-replication of the above-mentioned recombinant expression vector and the nucleic acid carried by it can be effectively expressed. Preferably, the said 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 a host cell can obtain the preferred recombinant expression transformant of the present invention. Among them, the said transformation method is a conventional transformation method in the art, preferably chemical transformation method, heat shock method or electroporation method.

[0202] Preparation of Antibodies

[0203] The sequence of the DNA molecule of the antibody or its fragment of the present invention can be obtained by conventional techniques, such as methods like PCR amplification or screening of genomic libraries. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0204] Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually to clone it into a vector, then transfer it into cells, and then separate the relevant sequence from the proliferated host cells by conventional methods.

[0205] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Generally, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them.

[0206] Currently, it is already possible to completely obtain the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention through chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.

[0207] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0208] The host cell can be a prokaryotic cell, such as a bacterial cell; or 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, HEK-293 cells.

[0209] Generally, the transformed host cells are cultured under conditions suitable for the expression of the antibody of the present invention. Then the antibody of the present invention is purified by 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 and other conventional separation and purification means well-known to those skilled in the art.

[0210] The obtained monoclonal antibodies can be identified by conventional means. 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 of Munson et al., Anal. Biochem., 107:220 (1980).

[0211] The antibodies of the present invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If needed, the recombinant protein can be separated and purified by various separation methods using its 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 renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0212] Antibody-drug conjugate (ADC)

[0213] As used herein, the terms "antibody-drug conjugate", "ADC", and "antibody-conjugated drug" are used interchangeably.

[0214] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0215] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, the antibody being conjugated to the effector molecule, preferably by chemical conjugation. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0216] The antibody of the present invention and the effector molecule can be conjugated through a coupling agent. Examples of the coupling agent can be any one or several of a non-selective coupling agent, a coupling agent using carboxyl groups, a peptide chain, and a coupling agent using disulfide bonds. The non-selective coupling agent is a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent using carboxyl groups can be any one or several of cis-aconitic anhydride-based coupling agents (such as cis-aconitic anhydride) and acylhydrazone-based coupling agents (the coupling site is acylhydrazone).

[0217] Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly connected to the antibody through a linker.

[0218] Antibodies can be conjugated with drugs to form antibody-drug conjugates (ADCs). Typically, an ADC comprises a linker positioned between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers typically degrade readily in the intracellular environment, e.g., at the target site, such that the drug is released from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, which include peptide-based linkers that can be degraded by intracellular proteases (e.g., lysosomal or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. Peptide-based 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 a 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 proteolytically cleaved.

[0219] Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and the attachment is effected through the reactive groups. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodo-, bromo- or chloro-substituted), haloesters (e.g., iodo-, bromo- or chloro-substituted), halomethyl ketones (e.g., iodo-, bromo- or chloro-substituted), benzyl halides (e.g., iodo-, bromo- or chloro-substituted), vinyl sulfones, pyridyl disulfides, mercury derivatives such as 3,6-di-(mercurimethyl) dioxane with the counterion being acetate, chloride or nitrate, and polymethylene dimethyl thiosulfonate. The linker can include, for example, a maleimide attached to the antibody through a succinimide.

[0220] The drug can be any cytotoxic, cell growth-inhibiting or immunosuppressive drug. In an embodiment, the linker connects the antibody and the drug, and the drug has a functional group capable of bonding to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group capable of bonding to the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0221] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. Examples of particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Exemplary cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines, maytansinoids (such as DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0222] In the present invention, the drug-linker can be used to form an ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0223] Typically, functional groups on the linker are selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As non-limiting examples, azide-based moieties can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker through a 1,3-dipolar cycloaddition between the azide and the alkyne. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (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 conjugation strategies, such as those described in Bioconjugate Techniques, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for either the linker or the drug.

[0224] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0225] In certain embodiments, the method of the present invention comprises: combining an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: combining the antibody-linker conjugate with a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody through the linker.

[0226] In some embodiments, the antibody-drug conjugate ADC is represented by the following formula:

[0227]

[0228] Wherein:

[0229] Ab is an antibody,

[0230] LU is a linker;

[0231] D is a drug;

[0232] And the subscript p is a value selected from 1 to 8.

[0233] Use

[0234] The present invention also provides the use of the antibody, recombinant protein, chimeric antigen receptor (CAR) construct and / or immune cell of the present invention, for example, for preparing a diagnostic agent or preparing a drug.

[0235] Preferably, the drug is a drug for preventing and / or treating diseases associated with abnormal p-Tau217 protein expression or function.

[0236] Preferably, the diagnostic agent is used for diagnosing diseases associated with p-Tau217 protein expression, such as Alzheimer's disease.

[0237] In the present invention, the diseases associated with abnormal p-Tau217 protein expression or function are conventional diseases in the art associated with abnormal p-Tau217 protein expression or function. Preferably, the diseases associated with abnormal p-Tau217 protein expression or function are Alzheimer's disease.

[0238] Detection uses and kits

[0239] The antibody of the present invention can be used for detection applications, such as for detecting samples to provide diagnostic information.

[0240] In the present invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" used in the present invention shall include all types of biopsies known to those skilled in the art. Therefore, the biopsies used in the present invention can include, for example, excised samples of tumors, tissue samples prepared by endoscopic methods or puncture or needle biopsies of organs.

[0241] The samples used in the present invention include fixed or preserved cell or tissue samples.

[0242] The present invention also provides a kit containing the antibody (or its fragment) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, an instruction manual, a buffer, etc. In the preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0243] The kit of the present invention is used for detecting p-Tau217 protein to diagnose Alzheimer's disease.

[0244] Pharmaceutical compositions

[0245] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or the corresponding immune cells, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the substances being formulated and the disease to be treated.

[0246] The prepared pharmaceutical composition can be administered through conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the preferred route of administration of the pharmaceutical composition of the present invention is injection or oral administration. The injection administration preferably includes routes such as intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is various conventional dosage forms in the art, preferably in solid, semi-solid, or liquid form, can be aqueous solution, non-aqueous solution, or suspension, and more preferably tablets, capsules, granules, injections, or infusions, etc.

[0247] The antibody of the present invention can also be expressed intracellularly by a nucleotide sequence for cell therapy. For example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T), etc.

[0248] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and / or treating diseases related to abnormal p-Tau217 protein expression or function.

[0249] The pharmaceutical composition of the present invention can be directly used to bind to p-Tau217 protein molecules, and thus can be used to prevent and treat diseases such as tumors.

[0250] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should match the administration method. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0251] In the present invention, preferably, the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiological or pharmaceutically acceptable drug excipient. The drug excipient is a conventional drug excipient in the art, and preferably includes pharmaceutically acceptable excipients, fillers, or diluents, etc. More preferably, the pharmaceutical composition includes 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of the pharmaceutical carrier, and the percentages are mass percentages of the pharmaceutical composition.

[0252] In the present invention, preferably, the dosage of the pharmaceutical composition is an effective amount, and the effective amount is an amount capable of alleviating or delaying the progression of a disease, a degenerative or traumatic 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 results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using no more than routine experiments.

[0253] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally 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 dose should also consider factors such as the route of administration and the health status of the patient, which are all within the scope of the skills of a skilled physician.

[0254] The present invention provides the use of the above pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease 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.

[0255] The main advantages of the present invention include:

[0256] 1. The antibody of the present invention has good specificity and can specifically bind to p-Tau217 protein. Developing a detection method for p-Tau217 protein based on the antibody of the present invention can effectively avoid false positive results and provide the accuracy of the detection method.

[0257] 2. The detection method for p-Tau217 protein developed using the antibody of the present invention has high sensitivity and a good linear relationship. It can detect low concentrations of p-Tau217 protein, which is beneficial for the early diagnosis of Alzheimer's disease.

[0258] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out 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 according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0259] Example 1 Preparation of Rabbit Monoclonal Antibody Against p-Tau217 Protein

[0260] Step 1: Antigen preparation

[0261] Select the Tau protein sites 208 - 227 as the p-Tau 217 polypeptide region, with the sequence:

[0262] SRSRTPSLP{pT}PPTREPKKVA (SEQ ID NO:33), where threonine at position 217 is phosphorylated;

[0263] Select the Tau protein sites 173 - 191 as the p-Tau 181 polypeptide region, with the sequence:

[0264] AKTPPAPK{pT}PPSSGEPPKS (SEQ ID NO:34), where threonine at position 181 is phosphorylated;

[0265] Select the Tau protein sites 208 - 227 as the non-phosphorylated 217 polypeptide region, with the sequence:

[0266] SRSRTPSLPTPPTREPKKVA (SEQ ID NO:33). When synthesizing the p-Tau 217 polypeptide, add the amino acid GGC at the C-terminus to link to the carrier protein KLH as the immunogen.

[0267] When synthesizing the p-Tau 217 polypeptide, add the amino acid GGC at the C-terminus to link to biotin as the positive screening antigen.

[0268] When synthesizing the non-phosphorylated Tau 217 polypeptide, add the amino acid GGC at the C-terminus to link to biotin as the negative screening antigen.

[0269] When synthesizing the p-Tau 181 polypeptide, add the amino acid GGC at the C-terminus to link to biotin as the negative screening antigen.

[0270] Step 2: Antigen immunization

[0271] Select healthy New Zealand white rabbits, and calculate the antigen dosage at 500 μg per rabbit per immunization. For the first immunization, mix the immunogen with an equal volume of Freund's complete adjuvant to make an emulsifier, and inject subcutaneously at multiple points. After a 2-week interval, take 500 μg of the immunogen and mix it with an equal volume of Freund's incomplete adjuvant to make an emulsifier, and inject subcutaneously at multiple points for two booster immunizations. After three immunizations, collect the rabbit peripheral blood samples, coat the plate with the p-Tau 217 polypeptide not conjugated to KLH, and determine the serum titer by ELISA method. Select the rabbits with high serum titer, inject 500 μg of the immunogen subcutaneously at multiple points for one booster immunization, and then take 15 mL of fresh rabbit peripheral blood into an anticoagulant tube.

[0272] Step 3: Isolation of rabbit peripheral blood PBMC cells

[0273] Dilute whole blood with an equal volume of whole blood and tissue diluent, and aliquot it into two 50 mL centrifuge tubes. Add 20 mL of separation solution to the two 50 mL centrifuge tubes, and gently layer the diluted blood on top of the separation solution, taking care to keep the interface between the two liquid layers clear. Centrifuge at 800×g for 30 min at room temperature using a horizontal rotor. After centrifugation, there will be a distinct layering: the top layer is the diluted plasma layer, the middle layer is the transparent separation solution layer, the white membrane layer between the plasma and the separation solution is the lymphocyte layer, and the bottom of the centrifuge tube contains red blood cells and granulocytes. Carefully aspirate the cells in the white membrane layer into a clean 15 mL centrifuge tube, and wash the cells in the white membrane layer 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 wash solution, and centrifuge at 250×g for 10 min. Repeat the washing once, then discard the supernatant and resuspend the cells in 1 mL of MACS buffer.

[0274] Step 4: Enrichment and activation of rabbit memory B cells

[0275] Sort rabbit memory B cells expressing IgG according to the rabbit memory B cell sorting protocol. Then centrifuge to collect the cells, resuspend the cells using rabbit memory B cell activation medium, and plate them in a 24-well plate. Incubate the cells in a 37°C, 5% CO2 incubator for 5 days, and add fresh rabbit memory B cell activation medium after 48 hours.

[0276] Step 5: Screening of rabbit single B cells

[0277] According to the single B cell screening protocol, transfer the activated plasma B cells into the instrument, and then incubate the positive antigen, negative antigen with the activated rabbit plasma B cells. Screen for rabbit plasma B cells that can secrete non-cross-reactive antibodies through the fluorescence signal of the instrument.

[0278] Step 6: Export rabbit single B cells and complete amplification of antibody variable region sequences

[0279] Transfer the screened rabbit single B cells into a 96-well plate, one cell per well, using software. Then, through a single-cell cDNA amplification kit, amplify to obtain the single B cell cDNA sequence, and then amplify the antibody heavy and light chain variable region sequences by PCR, and subsequently send them for sequencing.

[0280] Figure 1 Figure [ID] shows the agarose gel electrophoresis band pattern of the PCR products of the antibody variable region genes. The antibody pairs with successful sequencing are within the box in the figure. The sequencing results are shown in Table 1.

[0281] Table 1

[0282]

[0283]

[0284] Step 7: Recombinant expression of the antibody

[0285] The light and heavy chain sequences of the antibody were synthesized into the pcDNA3.4 expression vector by gene synthesis. The inserted restriction enzyme sites were EcoRI and HindIII. Then, the plasmid was transfected into Expi293 cells by transient transfection for expression. Specifically, the amino acid sequence of the antibody was first optimized for codons to adapt to 293 cells. Through gene synthesis, the sequence was synthesized into the vector. After synthesis, Escherichia coli DH5α competent cells were transformed. Then, positive clones were picked for plasmid extraction using the Tiangen plasmid miniprep kit. Finally, the light and heavy chain plasmids were transfected into Expi293F cells by transient transfection for antibody expression.

[0286] Step 8: Purification of the antibody

[0287] The cell supernatant was collected 5 - 7 days after transfection to obtain the recombinant antibody by purification. Specifically, before purification, the cell supernatant was centrifuged at 4000 rmp for 20 minutes, then filtered through a polypropylene membrane with a pore size of 0.22 μm, and then the cell supernatant was purified using an AKTA Pure purifier and a protein A affinity purification column.

[0288] After purification, SDS-PAGE electrophoresis combined with staining analysis was used to detect and verify the purity and integrity of the antibody sample.

[0289] Figure 2 The SDS-PAGE results after antibody purification are shown. NR is non-reducing electrophoresis staining, and R is reducing electrophoresis staining.

[0290] Example 2 Verification of the specificity of the p-Tau217 monoclonal antibody

[0291] 2.1 Method

[0292] Step 1: Antigen coating and blocking

[0293] Commercially available full-length p-Tau217 antigen (recombinant antigen), full-length p-Tau181 antigen (recombinant antigen), and non-phosphorylated total-Tau antigen (recombinant antigen) were respectively diluted to a concentration of 1.5 μg / mL with CB buffer and coated into a colorless transparent ELISA plate at 100 μL per well. Then, the ELISA plate was blocked overnight at 4°C with a blocking solution containing BSA.

[0294] Step 2: Indirect method to test the specificity of the p-Tau217 monoclonal antibody

[0295] After sealing, the plate is shaken off the liquid in the plate and the residual liquid is patted dry for later use. Dilute the p-Tau217 monoclonal antibody with PBS, and the dilution gradients are 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, 0 ng / mL. Then add them into the ELISA plates coated with three antigens respectively, 100 μL per well, react at 37 °C for 30 min, shake off the liquid in the plate and pat dry the residual liquid. Add 300 μL of 1X washing solution to each well with a plate washer, wash 5 times, shake off the liquid in the plate and pat dry the residual liquid. Add 100 μL of enzyme-labeled goat anti-rabbit secondary antibody at 0.5 μg / mL to each well. React at 37 °C for 30 min. Shake off the liquid in the plate and pat dry the residual liquid. Add 300 μL of 1X washing solution to each well with a plate washer, wash 5 times, shake off the liquid in the plate and pat dry the residual liquid. Add 100 μL of chromogenic solution to each well and react at 37 °C for about 15 min. Add 50 μL of stop solution to each well. Measure the OD value within 15 min after adding the stop solution, and the detection wavelength is 450 nm.

[0296] 2.2 Results

[0297] Table 1

[0298]

[0299] Table 2

[0300]

[0301] Table 3

[0302]

[0303] As shown in Tables 1-3, the 3 selected p-Tau217 monoclonal antibodies have very strong specificity for the p-Tau217 antigen on the market and have no cross-reaction with non-phosphorylated total-Tau217 antigen and p-Tau181.

[0304] Example 3 Affinity Test

[0305] Step 1: Pre-enrich the antigen on the CM5 chip: Dilute the full-length p-Tau217 antigen (recombinant antigen) with sodium acetate solutions at pH 5.0, pH 4.5, and pH 4.0 to 20 μg / mL respectively, and then inject them into the chip on the machine to detect the change in the RU value of the instrument. According to the results, select the sodium acetate solution at pH 4.5 to dilute the antigen to 10 μg / mL for the next step of chip antigen coupling.

[0306] Step 2: Conjugation of antigen: The commercially available recombinant p-Tau217 antigen (absea, PC006) was conjugated to the chip. First, the antigen was diluted to 10 μg / mL with a sodium acetate solution at pH 4.5. 200 μL was taken and loaded onto the machine for conjugation with the chip. After waiting for 30 minutes, the instrument automatically completed the conjugation of the antigen and the chip.

[0307] Step 3: Multi-cycle kinetic assay analysis: Three selected 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, 0 ng / mL. Detection was performed on the SPR detection instrument (cytiva, Biacore T200) sequentially from the lowest concentration. By analyzing the antigen-antibody binding constant Ka and dissociation constant Kd, the antibody affinity constant KD was finally determined.

[0308] The measurement results are shown in Table 4.

[0309] Table 4

[0310] Antibody Name ka kd KD Antibody 1 1.286E+04 2.852E-07 2.217E-11 Antibody 2 3.452E+04 4.568E-07 1.323E-11 Antibody 3 5.292E+5 1.907E-5 3.603E-11

[0311] As shown in Table 4, the affinities of the p-Tau217 rabbit monoclonal antibodies screened in the present invention all reached a relatively high level.

[0312] Example 4 Application of p-Tau217 Monoclonal Antibody in the Diagnosis of Alzheimer's Disease

[0313] Step 1: HRP-labeling of p-Tau217 monoclonal antibody

[0314] Antibody 1 was labeled with HRP enzyme. The specific method was to first activate the HRP enzyme with NaIO4, then perform a conjugation reaction between the activated HRP enzyme and the antibody in proportion, and add NaBH4 to reduce the sugar to stabilize the Schiff base (NH4)2SO4, so that the required enzyme precipitated to obtain the required enzyme.

[0315] Step 2: Coating of Tau protein antibody onto ELISA plate

[0316] The commercially available total-Tau protein antibody was used and diluted to a concentration of 1.5 μg / mL with CB buffer. 100 μL per well was coated onto a colorless transparent ELISA plate. Then, the ELISA plate was blocked overnight at 4°C with a blocking solution containing BSA.

[0317] The HRP-labeled p-Tau217 monoclonal antibody and the commercially available total-Tau protein antibody were combined into an ELISA kit.

[0318] Step 3: Sensitivity Verification of ELISA Kit

[0319] Collect cerebrospinal fluid samples from clinical AD patients, send the collected cerebrospinal fluid samples to the simoa platform for value determination. After the value determination is completed, gradient dilute the clinical samples with sample diluent, and the dilution concentrations are shown in Table 5.

[0320] Table 5

[0321] Sample Number P-Tau217 Concentration (pg / mL) 1 0.00 2 0.42 3 0.80 4 1.59 5 3.19 6 6.37 7 12.75 8 25.49 9 50.98 10 101.96

[0322] Add 100 μL of clinical samples with different concentrations to the ELISA plate coated with total-Tau protein antibody, and react at 37 °C for 60 min. Discard the liquid in the plate and pat dry the residual liquid. Add 300 μL of 1X washing solution to each well with a plate washer, wash 5 times, discard the liquid in the plate and pat dry the residual liquid.

[0323] Then add 100 μL of HRP-labeled antibody 1 diluted to a certain concentration to each well. React at 37 °C for 30 min. Discard the liquid in the plate and pat dry the residual liquid. Add 300 μL of 1X washing solution to each well with a plate washer, wash 5 times, discard the liquid in the plate and pat dry the residual liquid. Add 100 μL of chromogenic solution to each well and react at 37 °C for about 15 min. Then add 50 μL of stop solution to each well. Measure the OD value within 15 min after adding the stop solution, and the detection wavelength is 450 nm.

[0324] The detection results are shown in Table 6.

[0325] Table 6

[0326] Concentration pg / mL OD450 0.00 0.050 0.41 0.067 0.80 0.077 1.59 0.102 3.19 0.159 6.37 0.275 12.75 0.591 25.49 0.913 50.98 1.59 101.96 2.82

[0327] As shown in Table 6 and Figure 3 shown, when the p-Tau217 monoclonal antibody is paired with the commercially available total-Tau protein antibody, it shows a good linear relationship in the concentration range of 1.0 pg / mL to 100 pg / mL in enzyme-linked immunosorbent assay (ELISA), and its correlation coefficient (R 2 ) is 0.9924. That is, when the p-Tau217 monoclonal antibody screened in the present invention is paired with the total-Tau protein antibody with higher affinity selected in the market, it can be used for the diagnosis of Alzheimer's disease.

[0328] In addition to its application in the development of ELISA methodology, this antibody is also applicable to other diagnostic platforms, including chemiluminescence, fluorescence immunochromatography, etc. Therefore, the p-Tau217 monoclonal antibody of the present invention has significant potential in assisting the diagnosis of Alzheimer's disease.

[0329] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An antibody or an antibody binding fragment thereof targeting p-Tau217 protein, characterized in that: 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: (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 comprises 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, characterized in that The recombinant protein has: (i) the antibody or antigen-binding fragment thereof according to claim 1; and (ii) optionally a tag sequence to facilitate expression and / or purification.

3. A chimeric antigen receptor CAR, characterized in that: 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 complementary determining regions CDR: (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 comprises 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, characterized in that The polynucleotide encodes the antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 2, or the chimeric antigen receptor CAR according to claim 3.

5. A carrier, characterized in that The vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5 or the exogenous polynucleotide according to claim 4 is integrated into its genome.

7. A method for preparing CAR-NK cells or CAR-T cells, characterized in that: The CAR-NK cell or CAR-T cell expresses the chimeric antigen receptor according to claim 3, comprising the following steps: The polynucleotide according to claim 4 or the vector according to claim 5 is transduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

8. A method for detecting p-Tau217 protein in a sample for non-diagnostic and non-therapeutic purposes in vitro, the method comprising the steps of: (1) contacting a sample with the antibody or antigen-binding fragment thereof according to claim 1, or the recombinant protein according to claim 2; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of p-Tau217 protein in the sample.

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

10. A use of the antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 2, the chimeric antigen receptor according to claim 3, the polynucleotide according to claim 4, the vector according to claim 5, the host cell according to claim 6 or the immunoconjugate according to claim 9, characterized in that: (a) preparing a detection reagent or a kit; and / or (b) preparing a drug or preparation for preventing and / or treating a p-Tau217 protein-related disease, wherein the p-Tau217 protein-related disease is Alzheimer's disease.

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