Anti-p-Tau181 protein monoclonal antibody as well as preparation method and application thereof

Abstract

The invention provides a monoclonal antibody of a p-Tau181 protein and an application of the monoclonal antibody. Specifically, the invention provides an antibody targeting p-Tau181 protein or an antigen binding fragment of the antibody. The antibody provided by the invention can specifically recognize and bind the p-Tau181 protein, does not recognize p-Tau217 and non-phosphorylated Tau protein, and can be used for preparing a detection preparation or a kit for diagnosing diseases related to the p-Tau181 protein, such as Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-p-Tau181 protein monoclonal antibody and a preparation method and application thereof. 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, Tau protein is overphosphorylated to form neurofibrillary tangles (NFTs). Among them, phosphorylated tau protein 181 site (p-Tau181) is considered to be a very sensitive and specific biomarker for early AD. Therefore, the development of antibodies that can specifically recognize p-Tau181 is of great significance for the early diagnosis of AD.

[0003] Existing technologies mainly use 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 use them for the diagnosis of AD. However, these antibodies may have certain limitations in specificity and sensitivity because they may also cross-react with non-phosphorylated tau proteins 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 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 antibodies.

[0005] Therefore, there is an urgent need in the art for an antibody that specifically binds to the p-Tau181 protein. Summary of the invention

[0006] The purpose of the present invention is to provide an antibody that specifically binds to p-Tau181 protein and a preparation method and application thereof.

[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 p-Tau181 protein antibodies, and to provide pharmaceutical compositions comprising the above active ingredients.

[0008] Another object of the present invention is to provide a method for preventing and / or treating a disease in which the p-Tau181 protein is highly expressed, and a method for diagnosing a disease in which the p-Tau181 protein is highly expressed.

[0009] In a first aspect of the present invention, an antibody or an antibody binding fragment thereof targeting p-Tau181 protein is provided, 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 group:

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

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

[0012] (z2) the heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO:7, VH-CDR2 shown in SEQ ID NO:8, and VH-CDR3 shown in SEQ ID NO:3; and / or

[0013] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:9, VL-CDR2 shown in SEQ ID NO:10, and VL-CDR3 shown in SEQ ID NO:11; or

[0014] (z3) the heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO: 12, VH-CDR2 shown in SEQ ID NO: 13, and VH-CDR3 shown in SEQ ID NO: 3; and / or

[0015] 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:10, and VL-CDR3 shown in SEQ ID NO:11.

[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: 22; and / or

[0017] The antibody or antigen-binding fragment comprises a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23.

[0018] 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: 24; and / or

[0019] The antibody or antigen-binding fragment comprises a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25.

[0020] 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: 26; and / or

[0021] The antibody or antigen-binding fragment comprises a light chain variable region having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:27.

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

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

[0024] In another preferred example, 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 example, the heavy chain FR region comprises FR-H1 shown in SEQ ID NO:14, FR-H2 shown in SEQ ID NO:15, FR-H3 shown in SEQ ID NO:16, and FR-H4 shown in SEQ ID NO:17.

[0026] In another preferred example, the light chain FR region comprises FR-L1 shown in SEQ ID NO:18, FR-L2 shown in SEQ ID NO:19, FR-L3 shown in SEQ ID NO:20, and FR-L4 shown in SEQ ID NO:21.

[0027] In another preferred embodiment, the antibody or antigen-binding fragment thereof targeting p-Tau181 is selected from the following group: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2, mini antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond-stabilized Fv protein ("dsFv"), or a combination thereof.

[0028] In another preferred example, the antibody specifically binds to p-Tau181 protein or a protein derived therefrom.

[0029] In another preferred example, the antibody is capable of specifically binding to p-Tau181 proteins derived from rabbit, human, mouse and cynomolgus monkey.

[0030] In another preferred example, the light chain of the antibody further includes a light chain constant region.

[0031] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin, preferably of human or rabbit origin.

[0032] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0033] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin, preferably of human or rabbit origin.

[0034] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0035] In another preferred embodiment, the antibody is a monoclonal antibody.

[0036] In another preferred embodiment, the antibody includes a monospecific, bispecific, trispecific antibody or a multispecific antibody.

[0037] In a second aspect, the present invention provides a recombinant protein, wherein the recombinant protein comprises:

[0038] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0039] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0040] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0041] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0042] In another preferred embodiment, the recombinant protein is a fusion protein.

[0043] In another preferred embodiment, the fusion protein is a monospecific antibody (ie, a monospecific antibody against p-Tau181 protein), a bispecific antibody, or a multispecific antibody (eg, a trispecific antibody).

[0044] In another preferred embodiment, the bispecific antibody or multispecific antibody is not only against p-Tau181 protein, but also specifically binds to additional target antigens (such as other Alzheimer's disease antigens).

[0045] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0046] In another preferred embodiment, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused with the element (i).

[0047] In a third aspect, the present invention provides a chimeric antigen receptor CAR, wherein the antigen binding domain of the chimeric antigen receptor contains an antibody single-chain variable region sequence scFv targeting the p-Tau181 protein, and the heavy chain variable region and light chain variable region of the scFv include the following complementarity determining region CDR:

[0048] (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

[0049] 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

[0050] (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:3; and

[0051] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:9, VL-CDR2 shown in SEQ ID NO:10, and VL-CDR3 shown in SEQ ID NO:11; or

[0052] (z3) the heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO: 12, VH-CDR2 shown in SEQ ID NO: 13, and VH-CDR3 shown in SEQ ID NO: 3; and

[0053] 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:10, and VL-CDR3 shown in SEQ ID NO:11.

[0054] In a fourth aspect, the present invention provides a polynucleotide, which encodes 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.

[0055] In another preferred example, the polynucleotide comprises coding sequences encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof.

[0056] In another preferred example, the polynucleotide comprises the heavy chain variable region encoding sequence as shown in SEQ ID NO:5.

[0057] In another preferred example, the polynucleotide comprises the light chain variable region encoding sequence as shown in SEQ ID NO:12.

[0058] The fifth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the fourth aspect of the present invention.

[0059] 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.

[0060] In another preferred embodiment, the vector is a retroviral vector.

[0061] The sixth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the fifth aspect of the present invention or an exogenous polynucleotide described in the fourth aspect of the present invention is integrated into its genome.

[0062] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0063] In another preferred embodiment, the cell is a mammalian cell.

[0064] In another preferred embodiment, the cells are NK cells or T cells.

[0065] In another preferred embodiment, the host cell is an engineered immune cell.

[0066] In a seventh aspect, the present invention provides a method for preparing a CAR-NK cell or a CAR-T cell, wherein the CAR-NK cell or the CAR-T cell expresses the chimeric antigen receptor according to the third aspect of the present invention, comprising the following steps:

[0067] The polynucleotide described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention is transduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

[0068] In an eighth aspect, the present invention provides a pharmaceutical composition, which contains the antibody or antigen-binding fragment thereof 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, or the host cell described in the sixth aspect of the present invention, or a combination thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0069] In another preferred embodiment, the pharmaceutical composition is a preparation, preferably a liquid preparation.

[0070] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0071] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of 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, 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, or a combination thereof and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

[0072] In another preferred embodiment, the pharmaceutical composition is used to detect Alzheimer's disease.

[0073] The ninth aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0074] (a) an antibody portion, wherein the antibody portion is selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, or a combination thereof; and

[0075] (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.

[0076] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0077] In a tenth aspect, the present invention provides a use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the chimeric antigen receptor according to the third aspect of the present invention, the polynucleotide according to the fourth aspect of the present invention, the vector according to the fifth aspect of the present invention, the host cell according to the sixth aspect of the present invention, the pharmaceutical composition according to the eighth aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention, wherein the use comprises one or more uses selected from the following group:

[0078] (a) preparing a detection reagent or kit for detecting p-Tau181 protein; and / or

[0079] (b) for preparing a drug or preparation for preventing and / or treating a disease related to p-Tau181 protein.

[0080] In another preferred embodiment, the p-Tau181 protein-related disease is Alzheimer's disease.

[0081] In another preferred embodiment, the detection reagent or kit is used to detect the expression level of p-Tau181 protein, thereby diagnosing Alzheimer's disease.

[0082] In the eleventh aspect of the present invention, a method for detecting p-Tau181 protein in a sample in vitro (including diagnostic or non-diagnostic) is provided, the method comprising the steps of:

[0083] (1) contacting a sample with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention;

[0084] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of p-Tau181 protein in the sample.

[0085] In another preferred embodiment, the detection is for in vitro non-therapeutic and non-diagnostic purposes.

[0086] In another preferred embodiment, the method is a cell immunochemistry (Immunocytochemistry staning, ICC) detection method, or an immunohistochemistry (Immunohistochemistry IHC) detection method, or a whole cell ELISA detection method, or a cell lysate ELISA detection method.

[0087] In another preferred embodiment, the method is used to diagnose Alzheimer's disease.

[0088] In a twelfth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0089] (a) culturing the host cell according to the sixth aspect of the present invention under conditions suitable for expression;

[0090] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention.

[0091] In the thirteenth aspect of the present invention, a detection plate is provided, which comprises: 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.

[0092] In a fourteenth aspect of the present invention, a kit is provided, comprising:

[0093] (1) a first container, wherein the first container contains the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0094] (2) a second container, wherein the second container contains a secondary antibody against the antibody;

[0095] Alternatively, the kit contains the detection plate described in the thirteenth aspect of the present invention.

[0096] In the fifteenth aspect of the present invention, a method for treating a disease associated with abnormal expression or function of the p-Tau181 protein is provided, comprising administering to a subject in need of treatment an appropriate amount of the antibody or antigen-binding fragment thereof described in the first aspect of 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.

[0097] In another preferred embodiment, the disease associated with abnormal expression or function of p-Tau181 protein is Alzheimer's disease.

[0098] In the sixteenth aspect of the present invention, provided is the use of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the host cell as described in the sixth aspect of the present invention and / or the pharmaceutical composition as described in the eighth aspect of the present invention in the preparation of a medicament for treating diseases related to abnormal expression or function of the p-Tau181 protein.

[0099] In another preferred embodiment, the abnormal expression of p-Tau181 protein refers to overexpression of p-Tau181 protein.

[0100] In another preferred embodiment, the overexpression refers to the ratio of the expression level of p-Tau181 protein (F1) to the expression level under physiological conditions (F0) (ie, F1 / F0) being ≥1.2, preferably ≥1.5, and more preferably ≥2.00.

[0101] 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 embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 The agarose gel electrophoresis diagram of the PCR products of the variable region genes of the antibodies of the present invention is shown.

[0103] Figure 2 Shown is the SDS-PAGE electropherogram after antibody purification.

[0104] Figure 3 The results of antibody purity analysis of the antibody 1 of the present invention by SEC-HPLC are shown.

[0105] Figure 4 The results of antibody purity analysis of the antibody 2 of the present invention by SEC-HPLC are shown.

[0106] Figure 5 The results of antibody purity analysis of antibody 3 of the present invention by SEC-HPLC are shown.

[0107] Figure 6 The linear fitting results of ELISA test results are shown. DETAILED DESCRIPTION

[0108] After extensive and in-depth research, a large number of experiments and screening, the inventors unexpectedly discovered for the first time a class of antibodies targeting p-Tau181 protein. The antibodies of the present invention can specifically recognize and bind to p-Tau181 protein, and do not recognize proteins phosphorylated at other sites of Tau protein, such as p-Tau217, nor do they recognize non-phosphorylated Tau181 protein. On this basis, the present invention was completed.

[0109] Specifically, the present invention provides an antibody that binds to p-Tau181 protein and its application. The antibody or antigen-binding fragment thereof described in the present invention has one or more of the following properties: 1. It binds to p-Tau181 protein with high affinity and specificity; 2. It specifically recognizes p-Tau181 protein expressed on the cell surface; 3. The antibody of the present invention can be used to detect p-Tau181 protein. The present invention also provides a method for preparing the antibody and its application.

[0110] 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, wherein 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: 3, and the VH comprises the amino acid sequence of SEQ ID NO: 1.

[0111] In certain embodiments, the antibody or antigen-binding fragment thereof has one or more properties selected from the following group:

[0112] 1) Able to specifically recognize p-Tau181 protein expressed on the cell surface;

[0113] 2) The human-mouse chimeric antibody can bind to human, monkey and rabbit p-Tau181 proteins.

[0114] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and an immunoconjugate (ADC molecule).

[0115] the term

[0116] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, 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 belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0117] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., are understood to include the stated elements or components but do not exclude other elements or components.

[0118] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0119] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0120] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0121] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0122] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not occur.

[0123] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequences described herein and the sequences with which they are identical may 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%.

[0124] As used herein, the term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to 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 complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0125] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0126] As used herein, the term "light chain variable region" is used interchangeably with "VL."

[0127] As used herein, the term "antigen binding domain" and the like include any naturally occurring, enzymatically available, synthetic or genetically modified polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains can be used, for example, an antigen binding fragment of an antibody derived from a complete antibody molecule. Such DNA is known and / or easily available or can be synthesized from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries). The DNA can be sequenced and chemically or by using molecular biological techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable layout, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0128] 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 antibodies (e.g., independent complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0129] 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 generally comprise at least one CDR adjacent to or in line with one or more framework sequences. In an antigen binding fragment having a VH domain associated with a VL 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 a VH-VH, VH-VL or VL-VL dimer. Alternatively, the antigen binding domain can contain a monomeric VH or VL domain.

[0130] In a given antibody light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat based on antibody sequence variability (Kabat, E., et al., US 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 loop structural position.

[0131] It should be understood that the precise amino acid sequence boundaries of the CDRs in the present invention may optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise indicated, in the present invention, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering positions according to the Kabat numbering system.

[0132] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; 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. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0133] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial b-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0134] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (Cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form conjugates. The present invention also includes cell surface markers or antigens combined with the nanobodies or fragments thereof against p-Tau181.

[0135] As used herein, the terms "hypervariable region", "hypervariable region", "complementarity determining region" and "complementarity determining region (CDR)" are used interchangeably.

[0136] In a preferred embodiment of the present invention, the heavy chain variable region of the Nanobody or antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0137] In a preferred embodiment of the present invention, the heavy chain of the Nanobody or antibody comprises the above-mentioned heavy chain variable region and heavy chain constant region.

[0138] In the present invention, the terms "nanoantibodies of the present invention", "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, all referring to polypeptides that specifically bind to p-Tau181 protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

[0139] 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, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0140] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0141] The heavy chain variable regions of the nanobodies or antibodies of the invention are of particular interest because they are at least partially involved in binding to antigen. Thus, the invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0142] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0143] 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 substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0144] The antibody of the present invention refers to a polypeptide having p-Tau181 protein binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0145] 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 encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0146] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, 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.

[0147] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0148] Table A

[0149]

[0150]

[0151] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide 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 a coding strand or a non-coding strand.

[0152] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0153] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0154] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents 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 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0155] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0156] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0157] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0158] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0159] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. 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.

[0160] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0161] The obtained transformant 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 cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0162] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, 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 a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0163] The Nanobodies or antibodies of the invention can be used alone or in combination or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety or any combination of these.

[0164] 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 a detectable product.

[0165] Therapeutic agents that can be combined or coupled 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. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0166] p-Tau181 protein

[0167] p-Tau181 protein, that is, Tau protein phosphorylated at the 181st threonine position, is an important biomarker for neurodegenerative diseases, especially showing great potential in the diagnosis of Alzheimer's disease (AD).

[0168] At present, the detection of p-Tau181 protein is mainly carried out through blood testing. Compared with the traditional methods based on cerebrospinal fluid (CSF) or positron emission tomography (PET), blood testing has significant advantages such as less invasiveness, lower cost and easier operation.

[0169] The p-Tau181 protein showed 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 state of Alzheimer's disease, such as AβPET status, tau PET status, etc. In addition, the p-Tau181 protein can also be used as a supplement to cerebrospinal fluid and PET for participant selection in clinical trials of novel disease-modifying treatments.

[0170] Anti-p-Tau181 protein antibody

[0171] In the present invention, the terms "antibody of the present invention" and "p-Tau181 protein antibody" are used interchangeably, and both refer to an antibody comprising a heavy chain variable region and a light chain variable region selected from the following groups:

[0172] (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 / or

[0173] 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

[0174] (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 / or

[0175] The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:9, VL-CDR2 shown in SEQ ID NO:10, and VL-CDR3 shown in SEQ ID NO:11; or

[0176] (z3) the heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO: 12, VH-CDR2 shown in SEQ ID NO: 13, and VH-CDR3 shown in SEQ ID NO: 3; and / or

[0177] 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:10, and VL-CDR3 shown in SEQ ID NO:11.

[0178] Preferably, the antibody described herein 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 (scFv), a single-domain antibody (sdAb), and a single-domain antibody (Signle-domain antibody), as well as a monoclonal antibody or a polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be prepared by a variety of approaches 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 by hybridoma technology.

[0179] The full-length antibody protein is a conventional full-length antibody 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 and the human heavy chain constant region and the human light chain constant region constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

[0180] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0181] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0182] The single-chain antibody 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.

[0183] Wherein, the animal is preferably a mammal, such as a mouse.

[0184] The antibody of the present invention may be a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting p-Tau181 protein (eg, human p-Tau181 protein, mouse p-Tau181 protein, cynomolgus monkey p-Tau181 protein, or rabbit p-Tau181 protein).

[0185] 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%.

[0186] In the above content of the present invention, more preferably, the number of the added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0187] Recombinant protein

[0188] 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.

[0189] Preferably, the recombinant protein further comprises an antibody heavy chain constant region and / or an antibody light chain constant region, wherein the antibody heavy chain constant region is conventional in the art, preferably a rat antibody heavy chain constant region or a human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.

[0190] In a preferred embodiment, the recombinant protein includes the antibody of the present invention.

[0191] The recombinant protein 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 (scFv), a single-domain antibody (sdAb) and a single-domain antibody (Single-domainantibody), as well as a monoclonal antibody or a polyclonal antibody prepared from the above antibodies.

[0192] The single-chain antibody 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.

[0193] The antigen-antibody binding domain protein fragment 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').

[0194] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0195] The single-domain antibody is a conventional single-domain antibody in the art, which only includes the heavy chain variable region.

[0196] Wherein, the preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: obtaining by separation from an expression transformant that recombinantly expresses the protein or obtaining by artificially synthesizing a protein sequence. The separation from the expression transformant that recombinantly expresses the protein is preferably the following method: cloning a polynucleotide molecule encoding the protein and having a point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant, obtaining a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the recombinant protein.

[0197] Polynucleotide

[0198] The present invention also provides a polynucleotide encoding the above-mentioned antibody or recombinant protein of the present invention or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

[0199] The method for preparing the polynucleotide is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0200] 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 substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

[0201] Carrier

[0202] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0203] The recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned nucleic acid molecule. The vector preferably includes various plasmids, cosmids, phages or virus vectors, etc.

[0204] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector.

[0205] Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the above-mentioned recombinant expression vector is transformed into a host cell to obtain it. The host cell is any conventional host cell in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E.coli TG1 or E.coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody). The aforementioned recombinant expression plasmid is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0206] Antibody preparation

[0207] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0208] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0209] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0210] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0211] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0212] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

[0213] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0214] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the 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 the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0215] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, 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 sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0216] Antibody-drug conjugates (ADCs)

[0217] As used herein, the terms "antibody-drug conjugate," "ADC," and "antibody-drug conjugate" are used interchangeably.

[0218] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0219] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. 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.

[0220] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0221] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0222] Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0223] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0224] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0225] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), and vinca alkaloids.

[0226] In the present invention, drug-linkers can be used to form ADCs in one simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker reacts with a drug to form an ADC.

[0227] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. 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 attachment strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that when a complementary pair of reactive functional groups is selected for selective reaction of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0228] The present invention also provides a method for preparing ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0229] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0230] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0231]

[0232] in:

[0233] Ab is antibody,

[0234] LU is the connector;

[0235] D is for medicine;

[0236] And the subscript p is a value selected from 1 to 8.

[0237] application

[0238] The present invention also provides uses of the antibodies, recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for preparing diagnostic preparations or preparing drugs.

[0239] Preferably, the drug is a drug for preventing and / or treating diseases associated with abnormal expression or function of p-Tau181 protein.

[0240] Preferably, the diagnostic preparation is used to diagnose a disease associated with p-Tau181 protein expression, for example, Alzheimer's disease.

[0241] In the present invention, the disease associated with abnormal expression or function of p-Tau181 protein is a conventional disease associated with abnormal expression or function of p-Tau181 protein in the art. Preferably, the disease associated with abnormal expression or function of p-Tau181 protein is Alzheimer's disease.

[0242] Test Uses and Kits

[0243] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0244] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a resection sample of a tumor, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.

[0245] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0246] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0247] The kit of the present invention is used for detecting p-Tau181 protein, thereby diagnosing Alzheimer's disease.

[0248] Pharmaceutical composition

[0249] 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 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, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the disease to be treated.

[0250] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0251] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0252] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal expression or function of p-Tau181 protein.

[0253] The pharmaceutical composition of the present invention can be directly used to bind to p-Tau181 protein molecules, and thus can be used to prevent and treat diseases such as tumors.

[0254] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0255] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0256] In the present invention, preferably, the amount of the pharmaceutical composition administered is an effective amount, which is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part 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 experiments that do not exceed routine.

[0257] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0258] The present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease associated with abnormal expression or function of p-Tau181 protein. Preferably, the disease associated with abnormal expression or function of p-Tau181 protein is Alzheimer's disease.

[0259] The main advantages of the present invention include:

[0260] 1. The antibody of the present invention has good specificity and can specifically bind to the p-Tau181 protein. The p-Tau181 protein detection method developed based on the antibody of the present invention can effectively avoid false positive results and improve the accuracy of the detection method.

[0261] 2. The p-Tau181 protein detection method developed using the antibody of the present invention has high sensitivity and good linearity, and can detect low concentrations of p-Tau181 protein, which is beneficial for the early diagnosis of Alzheimer's disease.

[0262] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0263] Example 1 Preparation of rabbit monoclonal antibody against p-Tau181 protein

[0264] Step 1: Antigen preparation

[0265] The 173-191 sites of Tau protein were selected as the p-Tau 181 polypeptide region, and the sequence was:

[0266] AKTPPAPK{pT}PPSSGEPPKS (SEQ ID NO: 29), wherein the threonine at position 181 is phosphorylated;

[0267] The 208-227 sites of Tau protein were selected as the p-Tau 217 polypeptide region, and the sequence was:

[0268] SRSRTPSLP{pT}PPTREPKKVA (SEQ ID NO: 28), wherein the threonine at position 217 is phosphorylated;

[0269] The 173-191 sites of Tau protein were selected as the non-phosphorylated 181 polypeptide region, and the sequence was:

[0270] AKTPPAPKTPPSSGEPPKS (SEQ ID NO: 29), when synthesizing the p-Tau 181 polypeptide, the amino acid GGC was added to the C-terminus for connection to the carrier protein KLH as an immunogen.

[0271] When synthesizing the p-Tau 181 peptide, amino acids GGC are added to the C-terminus to connect biotin as a positive screening antigen.

[0272] When synthesizing the non-phosphorylated Tau 181 peptide, amino acids GGC are added to the C-terminus to connect biotin as a negative screening antigen.

[0273] When synthesizing the p-Tau217 peptide, amino acids GGC are added to the C-terminus to connect biotin as a negative screening antigen.

[0274] Step 2: Antigen Immunization

[0275] Healthy New Zealand white rabbits were selected, and the amount of antigen was calculated according to 500 μg per immunization. For the first immunization, the immunogen was mixed with an equal amount of Freund's complete adjuvant to make an emulsion, which was injected subcutaneously at multiple points. After an interval of 2 weeks, 500 μg of the immunogen was mixed with an equal amount of Freund's unadjuvant to make an emulsion, which was injected subcutaneously at multiple points, and boosted twice. After three immunizations, rabbit peripheral blood samples were collected, coated with p-Tau181 polypeptide not coupled to KLH, and the serum titer was determined by ELISA. Rabbits with high serum titers were selected, and 500 μg of the immunogen was injected subcutaneously at multiple points for booster immunization. After one booster immunization, 15 mL of fresh rabbit peripheral blood was collected in an anticoagulant tube.

[0276] Step 3: Isolation of PBMC cells from rabbit peripheral blood

[0277] Dilute whole blood with an equal volume of whole blood and tissue diluent, and divide into two 50mL centrifuge tubes. Add 20mL separation solution to two 50mL centrifuge tubes, and spread the diluted blood on the surface of the separation solution, making sure to keep the interface between the two liquid surfaces clear. Centrifuge at room temperature, 800×g in a horizontal rotor for 30min. After centrifugation, obvious stratification will appear: the top layer is the diluted plasma layer, the middle layer is the transparent separation solution layer, the white film layer between the plasma and the separation solution is the lymphocyte layer, and the bottom of the centrifuge tube is red blood cells and granulocytes. Carefully pipette the white film layer cells into a 15mL clean centrifuge tube and wash the white film layer cells with 10mL PBS. Centrifuge at 250g for 10min. Discard the supernatant, resuspend the cells with 5mL PBS or cell washing solution, and centrifuge at 250×g for 10min. Repeat the wash once, discard the supernatant, and resuspend the cells with 1mL MACS buffer.

[0278] Step 4: Enrichment and activation of rabbit memory B cells

[0279] Rabbit memory B cells expressing IgG were sorted according to the rabbit memory B cell sorting process. Cells were then collected by centrifugation, resuspended in rabbit memory B cell activation medium, and plated in 24-well plates. Cultured in a 37°C 5% CO2 incubator for 5 days, and fresh rabbit memory B cell activation medium was added after 48 hours.

[0280] Step 5: Screening of rabbit single B cells

[0281] According to the single B cell screening process, the activated plasma B cells are introduced into the instrument, and then the positive antigen and negative antigen are incubated with the activated rabbit plasma B cells. The rabbit plasma B cells that can secrete non-cross-reactive antibodies are screened out through the fluorescence signal of the instrument.

[0282] Step 6: Export rabbit single B cells to complete antibody variable region sequence amplification

[0283] The screened rabbit single B cells were introduced into a 96-well plate through the software, with one cell per well. The single B cell cDNA sequence was then amplified using a single cell cDNA amplification kit, and the antibody light and heavy chain variable region sequences were amplified by PCR and then sent for sequencing.

[0284] Figure 1 The agarose gel electrophoresis band diagram of the antibody variable region gene PCR product is shown in Table 1. The box in the figure shows the antibody pair that was successfully sequenced.

[0285] Table 1

[0286]

[0287]

[0288] Step 7: Recombinant expression of antibodies

[0289] The light and heavy chain sequences of the antibody pair were synthesized into the pcDNA3.4 expression vector, and the restriction sites were inserted as EcoRI and HindIII, and then the plasmid was transferred into Expi293 cells by transient transfection for expression. Specifically, the antibody amino acid sequence was first codon-optimized to adapt to 293 cells, and the sequence was synthesized into the vector by gene synthesis. After the synthesis, the E. coli DH5α competent cells were transformed, and then the positive clones were picked for plasmid extraction. The Tiangen plasmid extraction kit was used for plasmid extraction, and then the light chain and heavy chain plasmids were transferred into Expi293F cells by transient transfection for antibody expression.

[0290] Step 8: Antibody purification

[0291] The cell supernatant was collected 6 to 12 days after transfection and purified to obtain the recombinant antibody. Before purification, the cell supernatant was centrifuged at 4000 rpm for 20 minutes, then filtered through a polypropylene membrane with a pore size of 0.22 um, and then purified using an AKTA Pure purifier and a protein A affinity purification column.

[0292] After purification, the purity and integrity of the antibody samples were tested and verified by SDS-PAGE electrophoresis combined with staining analysis.

[0293] Figure 2 The SDS-PAGE results after antibody purification are shown in Figure 1. The lanes from left to right are lanes 1, 2, 3, and 4.

[0294] Table 1 shows the purity statistics of antibodies 1, 2 and 3.

[0295] Table 1. Antibody purity

[0296] name Lanes purity Antibody 1 1 97.10％ Antibody 2 2 95.30％ Antibody 3 3 95.20％ Marker 4 /

[0297] Figure 3 , 4 and 5 show the antibody purity analysis results of SEC-HPLC of antibodies 1, 2 and 3 of the present invention, respectively.

[0298] Example 2 Specificity verification of p-Tau181 monoclonal antibody

[0299] 2.1 Methods

[0300] Step 1: Antigen coating and blocking

[0301] The commercially available p-Tau181 full-length antigen (recombinant antigen), p-Tau217 full-length antigen (recombinant antigen), and non-phosphorylated total-Tau antigen (recombinant antigen) were diluted to a concentration of 1.5ug / mL with CB buffer and coated into a colorless transparent ELISA plate at 100ul per well. The ELISA plate was then blocked with a blocking solution containing BSA at 4°C overnight.

[0302] Step 2: Indirect method to test the specificity of p-Tau181 monoclonal antibody

[0303] After blocking, the plate was shaken off the liquid in the plate and the residual liquid was patted off for use. The p-Tau181 monoclonal antibody was diluted with PBS, and the dilution gradient was 1000ng / mL, 250ng / mL, 62.5ng / mL, 15.625ng / mL, 3.906ng / mL, 0.977ng / mL, 0.244ng / mL, 0ng / mL, and then added to the ELISA plates coated with the three antigens, 100ul per plate, reacted at 37℃ for 30min, the liquid in the plate was shaken off and the residual liquid was patted off, 300μL of 1X washing solution was added to each well of the plate washer, washed 5 times, the liquid in the plate was shaken off and the residual liquid was patted off. 100μL of 0.5μg / mL enzyme-labeled goat anti-rabbit secondary antibody was added to each well. Reacted at 37℃ for 30min. Shake off the liquid in the plate and tap off the residual liquid. Use a plate washer to add 300μL 1X washing solution to each well, wash 5 times, shake off the liquid in the plate and tap off the residual liquid. Add 100μL color development solution to each well and react at 37℃ for about 15 minutes. Add 50μL stop solution to each well. Measure the OD value within 15 minutes after adding the stop solution, and the detection wavelength is 450nm.

[0304] 2.2 Results

[0305] Table 2

[0306]

[0307]

[0308] Table 3

[0309]

[0310] Table 4

[0311]

[0312]

[0313] As shown in Tables 2-4, the three screened p-Tau181 monoclonal antibodies have very strong specificity for the p-Tau181 antigen on the market and have no cross-reaction with non-phosphorylated total-Tau217 antigen and p-Tau181.

[0314] Example 3 Affinity Test

[0315] Step 1: CM5 chip pre-enrichment of antigen: dilute the p-Tau181 full-length antigen (genetic recombinant antigen) to 20ug / mL with pH5.0, pH4.5, and pH4.0 sodium acetate solutions, respectively, and then inject them into the chip to detect the change in the instrument RU value. According to the results, select pH4.5 sodium acetate solution to dilute the antigen to 10ug / mL for the next step of chip coupling antigen.

[0316] Step 2: Antigen coupling: The commercially available p-Tau181 recombinant antigen (absea, PC006) was coupled to the chip. First, the antigen was diluted to 10ug / mL with a sodium acetate solution at pH 4.5, and 200ul was taken to couple with the chip. After waiting for 30 minutes, the instrument automatically completed the coupling of the antigen and the chip.

[0317] Step 3: Multi-cycle kinetic analysis: The three selected p-Tau181 rabbit monoclonal antibodies were diluted with HBS-EP+ buffer to a concentration gradient of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, and 0 ng / mL. The SPR detection instrument (cytiva, Biacore T200) was used for detection from the lowest concentration in turn, and the antibody affinity constant KD was finally determined by analyzing the antigen-antibody binding constant Ka and the dissociation constant Kd.

[0318] The measurement results are shown in Table 5.

[0319] Table 5. Antibody affinity test

[0320] Antibody Name ka kd KD <![CDATA[Full R 2 ]]> Antibody 1 5.74E+05 6.92E-05 1.21E-10 0.9969 Antibody 2 5.62E+05 5.20E-04 9.27E-10 0.9964 Antibody 3 4.62E+05 1.26E-04 2.73E-10 0.9959

[0321] As shown in Table 5, the affinity of the p-Tau181 rabbit monoclonal antibodies screened by the present invention all reached a relatively high level.

[0322] Example 4 Application of p-Tau181 monoclonal antibody in diagnosis of Alzheimer's disease

[0323] Step 1: HRP-labeled p-Tau181 monoclonal antibody

[0324] Antibody 1 is labeled with HRP enzyme. The specific method is to first activate the HRP enzyme with NaIO4, then couple the activated HRP enzyme with the antibody in proportion, add NaBH4 reducing sugar to stabilize Schiff base (NH4)2SO4, precipitate the required enzyme, and thus obtain the required enzyme.

[0325] Step 2: Coat Tau protein antibody onto ELISA plate

[0326] Use a commercially available total-Tau protein antibody, dilute it with CB buffer to a concentration of 1.5ug / mL, and coat 100ul per well on a colorless transparent ELISA plate. Then use a blocking solution containing BSA to block the ELISA plate at 4°C overnight.

[0327] HRP-labeled p-Tau181 monoclonal antibody and commercially available total-Tau protein antibody were combined into an ELISA kit.

[0328] Step 3: ELISA kit sensitivity verification

[0329] Cerebrospinal fluid samples were collected from clinical AD patients and sent to the simoa platform for determination. After determination, the clinical samples were gradient diluted with sample diluent. The dilution concentrations are shown in Table 5.

[0330] Table 6

[0331] Sample No. p-Tau181 concentration (pg / mL) 1 0.00 2 0.78 3 1.56 4 3.13 5 6.25 6 12.5 7 25 8 50 9 100 10 200

[0332] Add 100ul of clinical samples of different concentrations to the ELISA plate coated with total-Tau protein antibody and react at 37℃ for 60min. Shake off the liquid in the plate and tap off the residual liquid. Add 300μL of 1X washing solution to each well with a plate washer, wash 5 times, shake off the liquid in the plate and tap off the residual liquid.

[0333] Then add 100μL of HRP-labeled antibody diluted to a certain concentration to each well. React at 37℃ for 30min. Shake off the liquid in the plate and tap off the residual liquid. Use a plate washer to add 300μL 1X washing solution to each well, wash 5 times, shake off the liquid in the plate and tap off the residual liquid. Add 100μL of color development solution to each well and react at 37℃ for about 15min. Then add 50μL of stop solution to each well. Measure the OD value within 15min after adding the stop solution, and the detection wavelength is 450nm.

[0334] The test results are shown in Table 7.

[0335] Table 7

[0336]

[0337]

[0338] As shown in Table 7 and Figure 6 As shown in Figure 2, when the p-Tau181 monoclonal antibody was used in combination with a commercially available total-Tau protein antibody, it showed a good linear relationship in the enzyme-linked immunosorbent assay (ELISA) within the concentration range of 1.0 pg / mL to 100 pg / mL, and the correlation coefficient (R 2 ) is 0.998. That is, when the p-Tau181 monoclonal antibody screened by the present invention is paired with a total-Tau protein antibody with a higher affinity selected from the market, it can be used for the diagnosis of Alzheimer's disease.

[0339] In addition to the application in the development of ELISA methodology, the antibody can also be applied to other diagnostic platforms, including chemiluminescence, fluorescent immunochromatography, etc. Therefore, the p-Tau181 monoclonal antibody of the present invention has significant potential in assisting the diagnosis of Alzheimer's disease.

[0340] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An antibody or an antibody binding fragment thereof targeting p-Tau181 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 / or 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:3; and / or The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:9, VL-CDR2 shown in SEQ ID NO:10, and VL-CDR3 shown in SEQ ID NO:11; or (z3) the heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO: 12, VH-CDR2 shown in SEQ ID NO: 13, and VH-CDR3 shown in SEQ ID NO: 3; and / or 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:10, and VL-CDR3 shown in SEQ ID NO:

11.

2. A recombinant protein, characterized in that The recombinant protein comprises: (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-Tau181 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 / or 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:3; and / or The light chain variable region comprises the following three CDRs: VL-CDR1 shown in SEQ ID NO:9, VL-CDR2 shown in SEQ ID NO:10, and VL-CDR3 shown in SEQ ID NO:11; or (z3) the heavy chain variable region comprises the following three CDRs: VH-CDR1 shown in SEQ ID NO: 12, VH-CDR2 shown in SEQ ID NO: 13, and VH-CDR3 shown in SEQ ID NO: 3; and / or 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:10, and VL-CDR3 shown in SEQ ID NO:

11.

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, and the method comprises 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-Tau181 protein in a sample in vitro (including diagnostic or non-diagnostic), 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-Tau181 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: The use includes uses selected from the group consisting of: (a) preparing a detection reagent or kit for detecting p-Tau181 protein; and / or (b) for preparing a drug or preparation for preventing and / or treating a disease related to p-Tau181 protein.

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