Monoclonal antibody of anti-pT181 Tau protein and application thereof

Abstract

The invention discloses a monoclonal antibody of an anti-pT181Tau protein and an application of the monoclonal antibody of the anti-pT181Tau protein. The monoclonal antibody comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the heavy chain variable region VH, the HCDR2 and the HCDR3 are respectively shown as SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22. The monoclonal antibody provided by the invention has higher affinity and stronger specificity, and has a good effect on tissue staining.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular immunology, and particularly relates to a monoclonal antibody against pT181 Tau protein (T181 phosphorylated Tau protein) and its application. Background Art

[0002] Tau protein is one of the members of the microtubule-associated protein family, which can stabilize and regulate the assembly of microtubules and is encoded by a single-chain gene located on chromosome 17q21. Since the microtubule system is a skeletal component of neurons, Tau protein is widely expressed in the nervous system and participates in various cellular functions. Tau protein is closely related to the occurrence of neurodegenerative diseases and exerts its biological functions by activating different phosphorylation sites. In neurodegenerative diseases such as AD, the over-phosphorylation of tau severely reduces its binding ability to tubulin, making it insufficient to maintain the stability of microtubules. The tau dissociated from microtubules aggregates itself to form aggregates, which cross-link together to form neurofibrillary tangles (NFTs). These wrongly cross-linked NFTs will affect the normal physiological functions of neurons, increase their metabolic burden, and ultimately cause disorders of the nervous system function, damage protein transport, and lead to the death of a large number of neurons.

[0003] According to the dynamic biomarker hypothesis of AD diagnosis, amyloid pathological changes and neurodegenerative lesions occur before the clinical symptoms of AD appear; there are specific biomarkers reflecting the pathophysiological characteristics in each stage of AD clinical stages, including preclinical stage, mild cognitive impairment (MCI stage) and dementia stage. Currently, for the early diagnosis of AD, there are mainly two aspects of biomarkers with clinical significance and value:

[0004] 1. Cerebrospinal fluid biomarkers: such as Aβ, Tau protein, phosphorylated Tau protein (pTau) in cerebrospinal fluid;

[0005] 2. Neuroimaging biomarkers: Structural imaging, functional imaging, PET molecular imaging (mainly including Aβ imaging agents and Tau imaging agents). These two types of biomarkers have been verified by a large number of studies and have been confirmed to be able to stably and reliably reflect the earlier pathological changes of AD and have been included in the latest diagnostic criteria of the National Institute on Aging and the Alzheimer's Association (NIA / AA) and the International Working Group (IWG).

[0006] The anti - phosphorylated Tau protein antibody against T181 is a key raw material for the diagnosis of Alzheimer's disease. In China, this field is in its infancy, and there is still a certain gap compared with foreign products in terms of sensitivity and specificity. Currently, there are few domestic anti - abnormally phosphorylated Tau protein antibodies against T181. The few available ones are all polyclonal antibodies or mouse monoclonal antibodies, and no rabbit monoclonal antibodies have been seen. Moreover, the purchase of imported brands is costly. Therefore, there is a need to provide a monoclonal antibody with higher sensitivity and specificity. Summary of the Invention

[0007] To solve the problem of the lack of monoclonal antibodies with high sensitivity and specificity in the prior art, the present invention provides a monoclonal antibody against pT181 Tau protein and its application.

[0008] One of the technical solutions of the present invention is to provide a monoclonal antibody, which comprises a heavy - chain variable region VH and a light - chain variable region VL. The heavy - chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and the light - chain variable region VL comprises LCDR1, LCDR2 and LCDR3. Among them,

[0009] the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:17;

[0010] the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:18;

[0011] the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:19;

[0012] the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:20;

[0013] the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:21;

[0014] the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:22.

[0015] Among them, the amino acid sequence of SEQ ID NO:17 is X1YAMN, and the X1 is N or D;

[0016] the amino acid sequence of SEQ ID NO:18 is X2ISSSGKTYYAX3WAKG, the X2 is I or M, and the X3 is R or S;

[0017] the amino acid sequence of SEQ ID NO:19 is EGYAX4YYYGMDL, and the X4 is G or S;

[0018] The amino acid sequence of SEQ ID NO:20 is QX5SX6SX7X8NNX9X 10 LX 11 , where X5 is A or S; X6 is Q or K; X7 is L or V; X8 is Y or A; X9 is K or N; X 10 is N or W; X 11 is A or S;

[0019] The amino acid sequence of SEQ ID NO:21 is X 12 ASX 13 LAS, where X 12 is D or K; X 13 is T or S;

[0020] The amino acid sequence of SEQ ID NO:22 is QGTYX 14 SSGWYX 15 X 16 , where X14 is W or Y; X15 is Y or N; X16 is A or G.

[0021] In some preferred embodiments, the monoclonal antibody has an increase, deletion, and substitution of 1-2 amino acids on HCDR1, HCDR2, and / or HCDR3; and / or,

[0022] has an increase, deletion, and substitution of 1-2 amino acids on LCDR1, LCDR2, and / or LCDR3.

[0023] Preferably, in the monoclonal antibody:

[0024] The amino acid sequence of HCDR1 is as shown in SEQ ID NO:5;

[0025] The amino acid sequence of HCDR2 is as shown in SEQ ID NO:6;

[0026] The amino acid sequence of HCDR3 is as shown in SEQ ID NO:7;

[0027] The amino acid sequence of LCDR1 is as shown in SEQ ID NO:8;

[0028] The amino acid sequence of LCDR2 is as shown in SEQ ID NO:9;

[0029] The amino acid sequence of LCDR3 is as shown in SEQ ID NO:10; or,

[0030] The amino acid sequence of HCDR1 is as shown in SEQ ID NO:11;

[0031] The amino acid sequence of the HCDR2 is as shown in SEQ ID NO:12;

[0032] The amino acid sequence of the HCDR3 is as shown in SEQ ID NO:13;

[0033] The amino acid sequence of the LCDR1 is as shown in SEQ ID NO:14;

[0034] The amino acid sequence of the LCDR2 is as shown in SEQ ID NO:15;

[0035] The amino acid sequence of the LCDR3 is as shown in SEQ ID NO:16.

[0036] The antibody of the present invention has an affinity for binding of at least 8×10 -10 , up to 2×10 -12 M.

[0037] More preferably, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:1 or SEQ ID NO:3; the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:2 or SEQ ID NO:4;

[0038] Even more preferably, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:2; or,

[0039] the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:3 and the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:4.

[0040] In some more preferred embodiments, the monoclonal antibody includes one or more of a full-length antibody, an Fv such as a single-chain antibody (scFv), an antigen-binding fragment Fab, (Fab)2, and a bispecific antibody.

[0041] Preferably, the monoclonal antibody is a full-length antibody.

[0042] More preferably, the amino acid sequences of the heavy chain constant region and the light chain constant region of the full-length antibody are as shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.

[0043] The second technical solution of the present invention lies in providing an isolated nucleic acid, wherein the nucleic acid encodes the monoclonal antibody as described in any one of Technical Solutions 1 to 4.

[0044] A third technical solution of the present invention lies in providing a recombinant expression vector, wherein the recombinant expression vector contains the isolated nucleic acid as described in the second technical solution.

[0045] Preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

[0046] More preferably, the backbone of the recombinant expression vector is pcDNA3.1(+).

[0047] A fourth technical solution of the present invention lies in providing a transformant, wherein the recombinant expression vector as described in the third technical solution is contained in a host cell.

[0048] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0049] More preferably, the eukaryotic cell is a yeast cell or a mammalian cell.

[0050] Even more preferably, the host cell is a mammalian cell, such as HEK293F cell or CHO cell.

[0051] A fifth technical solution of the present invention lies in providing a method for preparing a monoclonal antibody targeting pT181 Tau protein, which comprises culturing the transformant as described in the fourth technical solution and obtaining the monoclonal antibody targeting pT181 Tau protein from the culture.

[0052] A sixth technical solution of the present invention lies in providing a kit, wherein the kit contains the monoclonal antibody as described in the first technical solution.

[0053] Preferably, the kit further contains one or more of the following reagents: BSA, PBS, pT181 Tau protein, horseradish peroxidase-conjugated goat anti-rabbit antibody, TMB and DAB chromogenic solution.

[0054] A seventh technical solution of the present invention lies in providing a pharmaceutical composition or a medicine box, wherein the pharmaceutical composition or the medicine box contains the monoclonal antibody as described in the first technical solution and a pharmaceutically acceptable carrier.

[0055] Preferably, the pharmaceutical composition or the medicine box further contains one or more of the group consisting of a hormone preparation, a targeted small molecule preparation, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule and a vaccine.

[0056] The eighth technical solution of the present invention lies in providing the use of the monoclonal antibody as described in Technical Solution 1, or the kit as described in Technical Solution 6, or the pharmaceutical composition or medicine box as described in Technical Solution 7 in the preparation of a medicament for detecting neurodegenerative diseases.

[0057] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0058] The ninth technical solution of the present invention lies in providing a method for non-diagnostic / therapeutic purposes of detecting pT181 Tau protein in neurodegenerative diseases, which comprises the step of using the monoclonal antibody as described in Technical Solution 1 or the kit as described in Technical Solution 6 for detection.

[0059] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0060] On the basis of conforming to common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0061] The reagents and raw materials used in the present invention are all commercially available.

[0062] The positive and progressive effects of the present invention are as follows:

[0063] The present invention provides a rabbit monoclonal antibody against pT181 Tau protein with high affinity. Compared with mouse antibodies and polyclonal antibodies, rabbit antibodies naturally have higher affinity (up to 10 -12 orders of magnitude) and stronger specificity, and achieve good results in tissue staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 It is the SDS-PAGE detection result of recombinant antibodies 46C5 and 46F1.

[0065] Figure 2 It is the ELISA affinity test of recombinant antibodies 46C5 and 46F1; where P1 represents phosphorylated polypeptide and P2 represents non-phosphorylated polypeptide.

[0066] Figure 3 It is the IHC detection result of recombinant antibody 46C5 on meningioma.

[0067] Figure 4 It is the IHC detection result of recombinant antibody 46F1 on glioma. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0068] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0069] The amino acid sequences of the CDRs listed in the present invention are all shown according to the Kabat definition rules. However, it is well known to those skilled in the art that in this field, there are various methods to define the CDRs of antibodies, such as Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the sequence variability of antibodies (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art should understand that unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or its region (such as the variable region) should be understood to cover the complementary determining regions defined by any of the above-known schemes described in the present invention.

[0070] Therefore, when referring to antibodies defined by the specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (such as different assignment system rules or combinations). Although the scope of protection claimed in the present invention is based on the sequences shown according to the Kabat definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0071] The term "variable region" refers to the domain in the antibody heavy chain involved in antibody binding to antigen. VH contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). Among them, the term "complementarity-determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains 3 CDR regions: HCDR1, HCDR2, and HCDR3. Each VH consists of 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.

[0072] The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a cell under conditions sufficient for the expression of the mRNA, protein, polypeptide, or peptide in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of the present disclosure are generally not naturally occurring. However, portions of the vector can be naturally occurring. The recombinant expression vectors of the present invention can contain any type of nucleotide, including but not limited to the following DNA and RNA: which can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and which can contain natural, non-natural, or altered nucleotides. The recombinant expression vector can contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In an exemplary aspect, the altered nucleotides or non-naturally occurring internucleotide linkages do not impede the transcription or replication of the vector.

[0073] The recombinant expression vectors of the present invention can be any suitable recombinant expression vectors capable of being used to transform or transfect a host cell with one or more genes or sequences of interest and preferably express the gene or sequence in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0074] As used herein, the term "host cell" refers to any type of cell that can contain the nucleic acids or vectors described herein. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or alga; or the host cell can be a prokaryotic cell, such as a bacterium or protozoan.

[0075] An expression vector can be transfected or introduced into a suitable host cell. This can be achieved by a variety of techniques, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transposons (Sleeping Beauty or PiggyBAC), gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultivated in a medium and screened for suitable activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and can be varied or optimized based on the methods known from the present specification and the prior art, depending on the specific expression vector and host cell used. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of the transfected host cells. The markers can, for example, provide prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics) or heavy metal (such as copper) resistance, etc. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers and termination signals.

[0076] The pharmaceutical composition of the present invention comprises a suitable pharmaceutically acceptable carrier such as pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used in the present invention, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, etc. The pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. A saline solution and aqueous dextrose, as well as glycerol solutions, can also be used as liquid carriers, especially for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skimmed milk, glycerol, propylene glycol, water, ethanol, etc. For the use and application of excipients, see also "Handbook of Pharmaceutical Excipients", 5th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of wetting agent or emulsifying agent, or pH buffering agent. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. The pharmaceutical preparation or pharmaceutical composition described in the present invention can be prepared by mixing the antibody or its antigen-binding fragment of the present invention with the desired purity with one or more optional pharmaceutical excipients (Remington’s Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)), preferably in the form of a freeze-dried preparation or an aqueous solution. The pharmaceutical composition or preparation of the present invention may also contain more than one active ingredient, which is required for the specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs or immunomodulators, etc. The active ingredients are suitably combined in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody or its antigen-binding fragment of the present invention, said matrix being in the form of a shaped article, such as a film or microcapsule.

[0077] The present invention selects the amino acid sequence near the T181 site, chemically synthesizes phosphorylated and non-phosphorylated Tau polypeptides containing the T181 site, conjugates the phosphorylated T181 polypeptide to the carrier protein KLH to make it immunogenic, and then immunizes New Zealand white rabbits. After the fourth immunization, biotin-labeled phosphorylated and non-phosphorylated T181 Tau polypeptides are used in combination with streptavidin as antigens, and the antiserum titer is detected by the enzyme-linked immunosorbent assay (ELISA) method. After the serum titer reaches a certain dilution, booster immunization is carried out, and the spleen is collected and ground. Total RNA is extracted from the spleen and reverse transcribed to synthesize the first-strand cDNA. By a set of primers matching the FR1 and FR4 of the variable regions of the heavy and light chains of rabbit antibodies, the variable region fragments (VH / Vκ) of the heavy and light chains of the immunized rabbits are amplified. By another set of primers for secondary amplification, a linker sequence with complementary overlapping regions is added to the variable fragments of the antibody heavy and light chains, and the VH fragment and the Vκ fragment are assembled into a single-chain antibody fragment (scFv) by overlap extension PCR (SOE PCR). After digesting the scFv fragment and the phage display vector with the same restriction endonuclease, they are ligated by gel extraction recovery. After cleaning the ligation product, it is electrotransformed into Escherichia coli TG1 to obtain a scFv phage display bacterial library. By culturing the bacterial library, helper phage M13K07 is added for superinfection, and the phage library is rescued by helper packaging, and the scFv fragment is displayed on the phage surface. The phosphorylated T181 Tau polypeptide is conjugated with biotin through the amino group and then captured by streptavidin-conjugated magnetic beads. The phage library is added. The phages displaying specific scFv fragments are captured because they can bind to the polypeptide, and the unbound phages are removed by rinsing. The phages are eluted from the magnetic beads, infect the TG1 host in the logarithmic growth phase, and after culturing, helper phage can be used again for superinfection and packaging rescue. After several rounds of screening, the TG1 bacterial solution infected with the screened phages is serially diluted, spread on a plate, and then monoclonal colonies are picked and cultured in a 96-well plate. Helper phage is added to package the phages of the monoclonal colonies, and the binding activity of the phages in the monoclonal supernatant to phosphorylated and non-phosphorylated T181 Tau polypeptides is detected by ELISA, and positive monoclonal clones 46C5 and 46F1 that specifically bind to the phosphorylated T181 site are obtained.

[0078] The candidate positive clones are subjected to sequencing analysis and constructed into the eukaryotic expression vector pcDNA3.1(+) containing the constant regions of rabbit antibody heavy and light chains and the IL2 signal peptide sequence to obtain a complete heavy and light chain expression vector. The heavy and light chain vector plasmids are co-transfected into HEK293F cells for expression, and the positive clone antibody protein is purified from the culture supernatant using Protein A. The binding specificity and affinity of the positive clone antibody protein to the pT181 phosphorylation site are verified by methods such as ELISA and IHC, and finally 2 antibodies that specifically bind to the phosphorylated T181 Tau polypeptide are obtained.

[0079] After the above antibody sequence was constructed into the expression vector pCDNA3.1 with the constant region added and transfected into HEK293F cells for expression, a recombinant monoclonal antibody was obtained after purification by protein A. It was verified by ELISA that it had a significantly strong binding force with the phosphorylated Tau protein polypeptide. At the same time, this antibody had an obvious staining effect in IHC experiments on brain tumor tissues such as glioma.

[0080] Example 1: Screening of the immunized rabbit antibody scFv display library

[0081] The full-length Tau protein has 441 amino acids. Since it is difficult to obtain an antibody specifically targeting phosphorylation at T181 by immunizing with the full-length protein, in the present invention, the phosphorylated polypeptide sequence PPAPKT(p)PPSSGC near the T181 position was synthesized, conjugated with KLH and outsourced to immunize New Zealand white rabbits. After 4 rounds of immunization, the serum titer reached 128,000. Rabbit spleen cells were ground and RNA was extracted to establish a cDNA library. The antibody variable region sequences in the library were amplified by the literature method and a phage display library was constructed for antibody screening. After two rounds of panning, 94 monoclonal clones were picked, and 36 positive clones were obtained. Two of the preferred ones were 46C5 and 46F1.

[0082] The amino acid sequence of the heavy chain variable region of 46C5 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of 46F1 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.4. The sequences underlined in the sequences are the antibody CDR regions.

[0083] 46C5 VH sequence: SEQ ID NO:1

[0084] QEQLVESGGRLVTPGTPLTLTCTVSGFSLS NYAMN WVRQAPGKGLEWIG MISSSGKTYYARWAKG RFTVSKTSTTVDLKITSPTTEDTATYFCAR EGYAGYYYGMDL WGPGTLVTVSS

[0085] 46C5 VL sequence: SEQ ID NO:2

[0086] DPMLTQTPSSSSAAVGGTVTINC QASQSLYNNKNLA WYQQKPGQPPKLLIY DASTLAS GVSSRFSGSGSGTQFTLTISGVQCDDAATYYC QGTYWSSGWYYA FGGGTKVEIK

[0087] 46F1 VH sequence: SEQ ID NO:3

[0088] QSVEESRGRLVTPGTPLTLTCTASGFSLS DYAMN WVRQAPGKGLEWIG IISSSGKTYYASWAKG RFTISKSSTTVDLKITSPTTEDTATYFCAR EGYASYYYGMDL WGPGTLVTVSS

[0089] 46F1 VL sequence: SEQ ID NO:4

[0090] DPVLTQTPSPVSAAVGGTVSISC QSSKSVANNNWLS WYQQKPGQPPKLLIY KASSLAS GVPSRFKGTGSGTQFTLTISEVQCDDAATYYC QGTYYSSGWYNG FGGGTKVEIK

[0091] Table 1 CDR sequences and sequence numbers of antibodies (determined by Kabat numbering rule)

[0092]

[0093]

[0094] The CDR sequences of the above two antibodies have high identity, so they are summarized as shown in Table 2.

[0095] Table 2 CDR sequences summarized from the above two antibodies

[0096]

[0097] Among them, X1 is N or D; X2 is I or M, X3 is R or S; X4 is G or S; X5 is A or S; X6 is Q or K; X7 is L or V; X8 is Y or A; X9 is K or N; X 10 is N or W; X 11 is A or S; X 12 is D or K; X 13 is T or S; X 14 is W or Y; X 15 is Y or N; X 16 is A or G.

[0098] Heavy chain constant region: SEQ ID NO:23

[0099] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0100] Light chain constant region: SEQ ID NO:24

[0101] PVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0102] Signal peptide sequence: SEQ ID NO:25

[0103] MYRMQLLSCIALSLALVTNS

[0104] Example 2: Expression and purification of positive cloned antibodies

[0105] Recombinant antibody expression: The heavy and light chain sequences of 46C5 and 46F1 were synthesized by Guangzhou Liuhe Gene Synthesis and optimized for codon sequences, and then constructed into the pCDNA3.1(+) vector. After obtaining the vector, the plasmid was extracted in large quantities, and then the heavy and light chain vectors were mixed at a molar ratio of 1:1. Take 30 mg of the vector, add 45 mL of OPM CD05 medium and 80 μL of 1 mg / mL PEI (POLYSCIENCE) solution, mix well, let stand at room temperature for half an hour, add it to 25 mL of HEK293F cells that are being cultured, put it in a shaker, culture at 37 °C, 8% CO2, 110 rpm for 7 days, add OPM 293 cell feed the next day, culture for 7 days, and centrifuge to collect the cell supernatant.

[0106] Recombinant antibody purification: 1 mL of protein A packing material (Sino Biological Inc.) was added to the received supernatant, and incubated at room temperature for half an hour. Then the packing material was taken out and loaded into an empty purification column, and 20 mL of PBS solution was added for rinsing. Then 10 mL of 100 mM glycine solution with pH 3.0 was added for elution, and 1 M Tris solution was added to the eluate to neutralize it to pH 7.0. The neutralized eluate was concentrated to 1 mL, the protein concentration was measured, and SDS-PAGE was run for purity identification. As can be seen from Figure 1 it that these two antibodies reached the electrophoretic purity standard, and no obvious other bands were seen except the main band.

[0107] Example 3: Determination of the affinity and specificity of positive clone antibodies

[0108] (1) The antigen phosphorylated polypeptide and the non-phosphorylated polypeptide with the same sequence were respectively conjugated with BSA, and 100 μL of the conjugated antigen with a concentration of 1 μg / mL BSA-polypeptide was coated on the enzyme strip overnight. Add 200 μL of PBST and shake for washing 3 times for 10 min each, and shake for washing 3 times with PBS.

[0109] (2) Add 200 μL of 1% BSA-PBS, and block at 37 °C for 2 h. Add 200 μL of PBST and shake for washing 3 × 10 min, and add 200 μL of PBS and shake for washing 3 × 10 min.

[0110] (3) After diluting the antibody at different concentrations, add 100 μL and incubate at 37 °C for 2 h. Add 200 μL of PBST and shake for washing 3 × 10 min, and add 200 μL of PBS and shake for washing 3 × 10 min.

[0111] (4) Add 100 μL of diluted commercial horseradish peroxidase-conjugated goat anti-rabbit antibody, and incubate at 37 °C for 1 h. Add 200 μL of PBST and shake for washing 3 × 10 min, and add 200 μL of PBS and shake for washing 3 × 10 min.

[0112] (5) Use 3,3′,5,5′-Tetramethylbenzidine microperoxidase substrate (TMB) as the substrate for color development for 10 min;

[0113] (6) After adding 50 μL of sulfuric acid with a concentration of 0.1 mol / L to terminate the reaction, measure its absorbance at 450 nm.

[0114] The results are as shown in Figure 2 it that it can be seen that both 46C5 and 46F1 only bind to the phosphorylated polypeptide and do not bind to the non-phosphorylated polypeptide, indicating that these two phosphorylated antibodies have good specificity. In addition, according to the ELISA results, it can be calculated that the affinities of the two antibodies are 2×10 -12 and 8×10 -10M, indicating that both antibodies have high affinity.

[0115] Example 4: IHC Application Assay of Positive Cloned Antibodies

[0116] Taking glioma and meningioma tissue sections as examples, the experimental process is as follows:

[0117] (1) Baking the slides: Put the slides into an incubator at 68 °C for about 90 minutes;

[0118] (2) Deparaffinization and dehydration: After baking the slides, put them into a deparaffinization jar, add xylene and soak for 3 times, 10 minutes each time, then soak in 100% alcohol for 5 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and rinse with water 3 times, 3 minutes each time;

[0119] (3) Antigen retrieval: Slowly put the slides into a pot with 0.01M citric acid buffer (pH 6.0), and continue to heat on an induction cooker for 15 minutes. Cool naturally at room temperature. After cooling to room temperature, take out the slides and rinse the slides with PBS buffer 3 times, 5 minutes each time;

[0120] (4) Blocking and inactivating endogenous peroxidase: Use a special oil-based pen for immunohistochemistry to draw a circle around the tissue specimen on the slide to prevent overflow during dropping. Add 3% H2O2 to the slide specimen after deparaffinization. After taking it out, slowly rinse the slide with the prepared PBS buffer 3 times, 5 minutes each time;

[0121] (5) Serum blocking: Gently shake off the water on the slide, place it on an incubation box, add normal goat serum working solution, incubate at room temperature for 10 minutes, and shake off;

[0122] (6) Adding primary antibody and secondary antibody: Drop 5 μg / mL of 46C5 and 46F1, and incubate overnight in a 4 °C refrigerator;

[0123] (7) Take it out the next morning and wait until it reaches room temperature. Rinse the slide with PBS buffer 3 times, 5 minutes each time. Then add HRP-labeled goat anti-rabbit secondary antibody working solution, incubate at room temperature for 10 minutes, take it out and rinse the slide with PBS buffer 3 times, 5 minutes each time;

[0124] (8) DAB / H2O2 reaction staining: Drop the newly prepared DAB chromogenic solution on the section;

[0125] (9) Counterstain with hematoxylin for 2 minutes, slowly rinse in clear water for 5 minutes, differentiate the tissue for 1 s, wash with clear water for 2 minutes, return to blue with PBS and then rinse with clear water for 2 minutes. Dehydrate 2 times each in 95% ethanol and absolute ethanol in ascending concentration, take out after 5 minutes each time;

[0126] (10) Immerse the glass slide in xylene solution twice, each time for 10 minutes, and then seal the slide with neutral balsam.

[0127] (11) Observe the staining of the specimen section under the microscope.

[0128] From the results of the staining of the section observed under the microscope, it can be seen that 46C5 ( Figure 3 ) and 46F1 ( Figure 4 ) both show obvious nerve cell staining for meningioma.

Claims

1. A monoclonal antibody, which comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3, characterized in that, The HCDR1 includes the amino acid sequence shown in SEQ ID NO:17; The HCDR2 includes the amino acid sequence shown in SEQ ID NO:18; The HCDR3 includes the amino acid sequence shown in SEQ ID NO:19; The LCDR1 includes the amino acid sequence shown in SEQ ID NO:20; The LCDR2 includes the amino acid sequence shown in SEQ ID NO:21; The LCDR3 includes the amino acid sequence shown in SEQ ID NO:

22.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody: has an addition, deletion and substitution of 1-2 amino acids on HCDR1, HCDR2 and / or HCDR3; and / or, has an addition, deletion and substitution of 1-2 amino acids on LCDR1, LCDR2 and / or LCDR3; Preferably, in the monoclonal antibody: The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:5; The amino acid sequence of the HCDR2 is as shown in SEQ ID NO:6; The amino acid sequence of the HCDR3 is as shown in SEQ ID NO:7; The amino acid sequence of the LCDR1 is as shown in SEQ ID NO:8; The amino acid sequence of the LCDR2 is as shown in SEQ ID NO:9; The amino acid sequence of the LCDR3 is as shown in SEQ ID NO:10; or, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:11; The amino acid sequence of the HCDR2 is as shown in SEQ ID NO:12; The amino acid sequence of the HCDR3 is as shown in SEQ ID NO:13; The amino acid sequence of the LCDR1 is as shown in SEQ ID NO:14; The amino acid sequence of the LCDR2 is as shown in SEQ ID NO:15; The amino acid sequence of the LCDR3 is as shown in SEQ ID NO:

16.

3. The monoclonal antibody according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:1 or SEQ ID NO:3; the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:2 or SEQ ID NO:4; Preferably, the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:2; or, The amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:3 and the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO:

4.

4. The monoclonal antibody according to claim 3, characterized in that, The monoclonal antibody includes one or more of a full-length antibody, Fv such as a single-chain antibody (scFv), an antigen-binding fragment Fab, (Fab)2, and a bispecific antibody; Preferably, the monoclonal antibody is a full-length antibody; More preferably, the amino acid sequences of the heavy chain constant region and the light chain constant region of the full-length antibody are as shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes the monoclonal antibody according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid according to claim 5; Preferably, the backbone of the recombinant expression vector is pcDNA3.1(+).

7. A transformant, characterized in that, Comprising the recombinant expression vector according to claim 6 in a host cell; Preferably, the host cell is a mammalian cell, such as HEK293F cell or CHO cell.

8. A method for preparing a monoclonal antibody targeting pT181 Tau protein, characterized in that, It comprises culturing the transformant according to claim 7 and obtaining a monoclonal antibody targeting pT181 Tau protein from the culture.

9. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the monoclonal antibody according to any one of claims 1 to 4.

10. A genetically modified cell, characterized in that, It includes the chimeric antigen receptor according to claim 9.

11. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent or a tag, and the monoclonal antibody according to any one of claims 1 to 4.

12. A kit, characterized in that, The kit comprises the monoclonal antibody according to any one of claims 1 - 4; Preferably, the kit further comprises one or more of the following reagents: BSA, PBS, pT181 Tau protein, horseradish peroxidase-conjugated goat anti-rabbit antibody, TMB and DAB chromogenic solution.

13. A pharmaceutical composition or a kit containing the same, characterized in that, The pharmaceutical composition or the kit containing the same comprises the monoclonal antibody according to any one of claims 1 to 4 or the genetically modified cell according to claim 10 or the antibody-drug conjugate according to claim 11, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition or the kit containing the same further contains one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

14. Use of the monoclonal antibody according to any one of claims 1-4, the genetically modified cell according to claim 10, the antibody-drug conjugate according to claim 11, or the pharmaceutical composition or the kit containing the same according to claim 13 in the preparation of a medicament for detecting neurodegenerative diseases; Preferably, the neurodegenerative disease is Alzheimer's disease.

15. A method for detecting pT181 Tau protein in neurodegenerative diseases, characterized in that, Comprising the step of detecting using the monoclonal antibody according to any one of claims 1 - 4 or the kit according to claim 9; Preferably, the method is for non-diagnostic / therapeutic purposes, and / or, the neurodegenerative disease is Alzheimer's disease.

16. A drug delivery device, characterized in that, The administration device comprises the monoclonal antibody according to any one of claims 1 - 4 or the genetically modified cell according to claim 10 or the antibody-drug conjugate according to claim 11 or the pharmaceutical composition or the kit containing the same according to claim 13; Preferably, the administration device further comprises a component for administering the monoclonal antibody, the chimeric antigen receptor, the antibody-drug conjugate, or the pharmaceutical composition or the kit containing the same to a subject, such as a syringe or an infusion device.

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