A p-Tau217 antibody and its humanized transformation and application

Abstract

The present invention relates to a p-Tau217 antibody and its humanized transformation and application. The amino acid sequence of the light chain variable region of the p-Tau217 antibody is: NILLTQSPASLAVSLGQRATISCRASESVD SYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK; the amino acid sequence of the heavy chain variable region of the p-Tau217 antibody is: EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQIPEKRLEWVASISSGGITFYPDSVKGRFTISTDNARNILYLQMSSLRSEDTAMYYCARGGDYGNFWFAYWGQGTLVTVSA. The complementary determining region of the p-Tau217 antibody is transplanted onto a human framework, and then the amino acids in the light chain framework region and / or the heavy chain framework region are reversely mutated to obtain a humanized p-Tau217 antibody. The p-Tau217 antibody and the humanized p-Tau217 antibody have good affinity with the p-Tau217 protein and have important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a p-Tau217 antibody and humanized transformation and application thereof. Background Art

[0002] Under normal physiological conditions, the microtubule-associated protein Tau binds to microtubules, promotes the assembly of microtubules, maintains the stability of microtubules, and plays an important role in the growth of axons and the transport of substances in neurons. In the neurodegenerative disease Alzheimer's disease (AD), Tau protein is abnormally modified, such as hyperphosphorylation, abnormal glycosylation, and nitration. This abnormally modified Tau protein loses its normal physiological function and eventually forms neurofibrillary tangles. p-Tau217 protein is phosphorylated Tau protein at the threonine 217 site. Its abnormal expression is closely related to the progression of AD. It has excellent diagnostic performance in distinguishing AD patients from other neurodegenerative diseases. In addition, it has the characteristics of strong operability, basically non-invasiveness, and low cost. It is considered to be one of the most promising peripheral blood AD early screening biomarkers in recent years. Therefore, the development of antibodies against p-Tau217 protein is of great significance for the diagnosis and treatment of AD.

[0003] In the prior art, the preparation of antibodies against specific antigens generally relies on the vaccination strategy of immune animals (such as mice). This process is mainly divided into the following steps: first, a suitable antigen is selected, such as p-Tau217; then, the antigen is injected into the animal to stimulate its immune system to produce specific antibodies against the target antigen; finally, with the help of hybridoma technology, hybridoma cells that can produce high-affinity antibodies are screened, and then the monoclonal antibodies are amplified. The significant advantage of this method is that a large number of antibodies can be obtained quickly. However, its limitations cannot be ignored, mainly reflected in the dependence on a large number of experimental animals and the cumbersome screening process. In addition, since these antibodies are obtained from animals, they may be incompatible with the human immune system, the so-called "xenoprotein reaction", which may weaken the therapeutic effect of the antibody and even induce adverse reactions. Furthermore, the prior art still has challenges in obtaining antibodies with high affinity and high specificity, and there are bottlenecks in the large-scale production of antibodies, which seriously hinders the clinical application of antibodies. Therefore, developing a humanized, high-affinity, high-specificity p-Tau217 antibody that is easy to scale up has become a core technical challenge that needs to be overcome in this research field. Summary of the invention

[0004] The purpose of the present invention is to provide a p-Tau217 antibody and its humanized transformation and application.

[0005] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a p-Tau217 antibody, the light chain variable region of the p-Tau217 antibody comprises: CDR L1 amino acid sequence: RASESVDSYGNSFLH, CDRL2 amino acid sequence: LTSNLES, CDRL3 amino acid sequence: QQNNEDPWT; the heavy chain variable region of the p-Tau217 antibody comprises: CDRH1 amino acid sequence: TYAM S, CDRH2 amino acid sequence: SISSGGITFYPDSVKG, CDRH3 amino acid sequence: GGDYGNFWFAY; further, the light chain variable region amino acid sequence of the p-Tau217 antibody is: NILLTQSPASLAVSLGQRATISC RASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTIDPVEAD DAATYYCQQNNEDPWTFGGGTKLEIK; the amino acid sequence of the heavy chain variable region of the p-Tau217 antibody is: EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQIPEKRLEWVASISSGGITFYPDSVKGRFTISTDNARNILYLQMSSLRSEDTAMYYCARGGDYGNFWFAYWGQGTLVTVSA.

[0006] The second aspect of the present invention provides a method for humanizing the above-mentioned p-Tau217 antibody, which comprises transplanting the complementary determining region of the p-Tau217 antibody onto a human framework, and then back-mutating the amino acids in the light chain framework region and / or the heavy chain framework region to restore the affinity of the humanized antibody; Furthermore, the back mutation in the light chain framework region is selected from any one or more of G72R, D1N, and M4L; the back mutation in the heavy chain framework region is selected from any one or more of R71T and G44R.

[0007] The third aspect of the present invention provides a humanized p-Tau217 antibody, which is obtained by the above method; further, the light chain variable region amino acid sequence of the humanized p-Tau217 antibody is selected from NIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK, DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK, DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK, any one of TFGGGTKVEIK, NIVLTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKL LIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK; the amino acid sequence of the heavy chain variable region of the humanized p-Tau217 antibody is selected from EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKRLEWVASISSGGITFYPDSVKGRFTISTDNAKNSLYLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS, EVQLVESGGGLVQPG GSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGITFYPDSVKGRFTISRDNAKNS Any of LYLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS, EVQLVESGGGLVQPGG SLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGITFYPDSVKGRFTISTDNAKNSL YLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS.

[0008] The fourth aspect of the present invention provides an isolated nucleic acid molecule, which encodes the above-mentioned humanized pT au217 antibody.

[0009] The fifth aspect of the present invention provides a method for preparing a humanized p-Tau217 antibody, which comprises cloning the above-mentioned nucleic acid molecule into a pcDNA3.4 expression vector containing a human IgG constant region to obtain a recombinant expression vector; transferring the recombinant expression vector into Expi293F cells to obtain recombinant cells; culturing the recombinant cells, and isolating and purifying the humanized p-Tau217 antibody from the culture.

[0010] The sixth aspect of the present invention provides the use of the above-mentioned p-Tau217 antibody and humanized p-Tau217 antibody in the preparation of a kit for auxiliary diagnosis of Alzheimer's disease.

[0011] The seventh aspect of the present invention provides the use of the above-mentioned p-Tau217 antibody and humanized p-Tau217 antibody in detecting p-Tau217 protein, and the application does not include application in disease diagnosis and treatment.

[0012] The significant advantages of the present invention are: 1. The present invention solves the problem that antibodies obtained in the prior art may be incompatible with the human immune system, namely the so-called "xenoprotein reaction". Through humanized design, the immunogenicity of the antibody is reduced and the safety of its clinical application is improved.

[0013] 2. The present invention solves the problem that the prior art still has challenges in obtaining antibodies with high affinity and high specificity. Humanized design is performed through computer software, which optimizes the affinity and specificity of the antibody and improves the clinical application effect of the antibody.

[0014] 3. The present invention solves the bottleneck problem in the existing technology in the large-scale production of antibodies. Through recombinant expression technology, large-scale production of antibodies is achieved to meet market demand.

[0015] 4. The present invention solves the problem that the prior art requires a large number of experimental animals and a cumbersome screening process. The antibody sequence is obtained by immunizing mice and screening hybridomas, which simplifies the antibody research and development process and improves the research and development efficiency.

[0016] 5. The present invention solves the deficiencies of the prior art in the research and application of p-Tau217 antibodies, and provides a new tool for the diagnosis and treatment of Alzheimer's disease by preparing humanized p-Tau217 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Linear fitting results of ELISA test results. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with examples. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0019] Example 1: Preparation method of mouse p-Tau217 antibody.

[0020] Step 1: Immunogen preparation The 210-224 sites of the total-Tau protein were intercepted as the p-Tau 217 antigen region, and the sequence was: SRTPSLP{pT}PPTREPK It was then sent to GenScript for peptide synthesis, during which the carrier protein BSA was coupled at the C-terminus and used as an immunogen for the next step of animal immunization.

[0021] Step 2: Immunization Healthy Balb / c mice were selected and injected for immunization. Specifically, the immunogen was injected into the mice at a dose of 100 μg / mouse, and booster immunization was performed every two weeks for a total of four immunizations. For the first immunization, the immunogen was mixed with an equal amount of Freund's complete adjuvant to form an emulsion, which was injected subcutaneously at multiple points. For the second and third immunizations, the immunogen was mixed with an equal amount of Freund's complete adjuvant to form an emulsion, which was injected subcutaneously at multiple points, and booster immunization was performed twice. Serum samples were collected after three immunizations, and the serum titer was determined by ELISA. Mice with high serum titers were selected, and the spleen was taken after a shock immunization with 100ug of immunogen injected intraperitoneally once. During this period, the immune response of the mice was closely observed to ensure that they could produce an effective immune response.

[0022] Step 3: Preparation of SP / 20 cells SP / 20 cells were revived two weeks before the completion of immunization. SP / 20 cells are a commonly used myeloma cell line. SP / 20 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at a dose of 3×10 per mouse. 7 SP / 20 cells were expanded and cultured in T75 square flasks for later use. The viability of SP / 20 cells was maintained at more than 95% before fusion, and the cells were washed 2 to 3 times with serum-free RPMI-1640 medium to remove serum.

[0023] Step 4: Preparation of mouse spleen cells After the mice were killed, they were soaked in 75% alcohol for 5 minutes, and then the spleen tissue was removed by aseptic operation. 10mL of serum-free culture medium (RPMI 1640) was drawn with a syringe. The forceps were held in the left hand, and the needle of the syringe was inserted into one end of the spleen with the right hand. The spleen cells were gently blown out, and the color of the spleen was visibly lighter. The obtained cell suspension was collected in a 50mL centrifuge tube, and the spleen was cut into blocks with scissors and placed on a 200-mesh sieve. Serum-free culture medium was added dropwise, and the piston rod of the syringe was used to grind through the sieve. This could be repeated 2 to 3 times. The cell suspension was collected in a 50mL centrifuge tube, washed twice with serum-free DMEM culture medium, counted, and resuspended with 20mL serum-free culture medium, placed in a 50mL polypropylene plastic centrifuge tube, and placed on ice.

[0024] Step 5: Fusion of mouse spleen cells and myeloma cells Mouse spleen cells were electrofused with SP / 20 cells. First, the prepared SP / 20 cells were mixed with mouse spleen cells in a ratio of 4:1, then washed twice with electrofusion solution, and then pulse fusion was performed according to the electrofusion procedure. After the fusion was completed, the fused cells were plated on a 96-well plate, and HAT was added for screening after 24 hours. After 5 to 7 days, the growth of the fused cells was observed, and the wells containing cell clusters were marked and indirect ELISA was performed to detect whether the cell culture supernatant contained specific antibodies.

[0025] Step 6: Antibody Screening The commercially available recombinant p-Tau217 antigen (Shanghai Fangyuan Biotechnology Co., Ltd., KSDR003-03) was coated onto the ELISA plate, and the positive wells were identified by ELISA. Specifically, the commercially available recombinant p-Tau217 antigen was coated on the ELISA plate at a dose of 1 μg / well and placed at 4°C overnight; the next day, it was washed with PBS, the liquid was dried, and the plate was washed 3 to 5 times; it was blocked with 2% BSA and incubated in a 37°C incubator for 2 hours; BSA was discarded, and the supernatant of the fusion well cells and the positive, negative, and blank controls were added to the ELISA plate, respectively, and the plates were incubated at 37°C for 60 minutes; the supernatant of the fusion well cells in the plate was discarded, the water was dried, the plate was washed with PBST, the liquid was dried, and the plate was washed 3 to 5 times; HRP-labeled sheep anti-mouse IgG secondary antibody was added to each well, and the plate was incubated at 37°C for 60 minutes; the secondary antibody in the plate was discarded, the plate was washed with PBST, the water was dried, and the plate was washed 3 to 5 times; TMB color development solution was added to each well, and the reaction was carried out at room temperature in the dark for 15 minutes, and then diluted sulfuric acid was added to terminate the reaction, and the OD was read as soon as possible using an ELISA reader. 450nm Value. 450nm Wells with values ​​3 times greater than the negative control were considered positive wells.

[0026] Step 7: Monoclonalization The selected hybridoma cells that can express high-affinity antibodies are monocloned twice, and each monoclonization is identified by ELISA, and finally a monoclonal hybridoma cell line is obtained. Specifically, the hybridoma cells are diluted by limiting dilution method and plated into 96-well plates at 0.7 cells / well (RPMI 1640 medium with 20% serum added in advance), and ELISA detection is performed after 5 to 7 days. A total of 2 rounds of monoclonization are performed.

[0027] Step 8: Obtaining antibody sequences.

[0028] The antibody variable region sequence was obtained using the hybridoma sequencing method. Specifically, the total RNA of the hybridoma cells was first extracted using a kit, and then reverse transcription was performed using a reverse transcription kit to synthesize cDNA. Next, PCR amplification was performed using mouse variable region amplification primers. After amplification, the product was gel-recovered and purified, cloned into a pEASY vector, and sequenced for the first generation. The kits used in this process were all from common manufacturers on the market.

[0029] The amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the mouse p-Tau217 antibody obtained by sequencing are as follows: VL amino acid sequence: NILLTQSPASLAVSLGQRATISCRASESVDSYGNSFLHWYQQKPGQPPKLL IYLTSNLESGVPDRFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK; wherein, the complementarity determining region (CDR) amino acid sequence: CDR1: RASESVDSYGNSFLH, CDR2: LTSNLE S, CDR3: QQNNEDPWT.

[0030] VH amino acid sequence: EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQIPEKRL EWVASISSGGITFYPDSVKGRFTISTDNARNILYLQMSSLRSEDTAMYYCARGGDYGNFWFA YWGQGTLVTVSA; among them, CDR amino acid sequence: CDR1: TYAMS, CDR2: SISSGGITFYPDSVKG, CDR3: GGDYGNFWFAY.

[0031] Implementation Example 2: Humanized Design The antibody humanization design function of WeMol (wemol.wecomput.com) was used to humanize the mouse p-Tau217 antibody through the CDR transplantation strategy. The heavy chain and light chain variable region germline genes with high homology to the chimeric antibody variable region were selected as templates by comparing the IMGT human antibody heavy and light chain variable region germline genes. The amino acid number and CDR region of the antibody were determined using the Kabat numbering system. The CDRs of the chimeric antibody were transplanted into the framework region (FR) of the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The applicability of the template was comprehensively scored by calculating the homology of the template gene, the frequency of occurrence in clinical and human, historical expression data, the isoelectric point of the transplanted sequence, and the PTM high-risk sites. The transplanted sequence of the high-scoring template was selected as the basis for the back mutation design. The humanized light chain templates of the chimeric antibody finally selected were IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV3-7*01 and IGHJ1*01.

[0032] Based on the predicted variable region structure, the effect of non-conservative amino acids in FR on CDR conformation was quantitatively evaluated, and the key amino acids with higher scores were backmutated to the corresponding amino acids of the chimeric antibody to maintain or improve the original affinity. Different backmutation combinations resulted in multiple humanized variants. The specific backmutation designs are shown in Table 1.

[0033] Table 1: Design of humanized antibody back mutation Note: Graft represents the implantation of chimeric antibody CDR into human germline template FR region sequence; G72R represents the mutation of G at position 72 of Graft to R; D1N represents the mutation of D at position 1 of Graft to N; M4L represents the mutation of M at position 4 of Graft to L; R71T represents the mutation of R at position 71 of Graft to T; G44R represents the mutation of G at position 44 of Graft to R. The numbering of the back-mutated amino acids is the natural sequence numbering.

[0034] The sequence of the humanized antibody variable region is as follows: VL1 amino acid sequence: DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPK LLIYLTSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEI K; VL2 amino acid sequence: DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPK LLIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEI K; VL3 amino acid sequence: NIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPK LLIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEI K; VL4 amino acid sequence: NIVLTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKL LIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK; VH1 amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGITFYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDYGNFWFAYW GQGTLVTVSS; VH2 amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGITFYPDSVKGRFTISTDNAKNSLYLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS; VH3 amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKRLEWVASISSGGITFYPDSVKGRFTISTDNAKNSLYLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS.

[0035] From the back mutation designs of the light chain and heavy chain variable regions of the above humanized antibodies, different light chain and heavy chain sequences were selected for cross-combination, and finally 12 humanized p-Tau217 antibodies were obtained. The amino acid sequences of the variable regions of the humanized p-Tau217 antibodies are as follows: Table 2: Amino acid sequences corresponding to the variable regions of humanized p-Tau217 antibodies F VH1 VH2 VH3 VL1 RY-1 RY-2 RY-3 VL2 RY-4 RY-5 RY-6 VL3 RY-7 RY-8 RY-9 VL4 RY-10 RY-11 RY-12 For example: the light chain variable region of the humanized p-Tau217 antibody RY-1 is VL1, and the heavy chain variable region is VH1; the light chain variable region of the humanized p-Tau217 antibody RY-9 is VL3, and the heavy chain variable region is VH3.

[0036] According to the Kabat numbering system, the results of the VH and VL sequence analysis of the above humanized p-Tau217 antibody are shown in Table 3.

[0037] Table 3: Kabat analysis results of humanized p-Tau217 antibody VH and VL sequences Variable region CDR1 CDR2 CDR3 VL1 RASESVDSYGNSFLH LTSNLES QNNEDPWT VL2 RASESVDSYGNSFLH LTSNLES QNNEDPWT VL3 RASESVDSYGNSFLH LTSNLES QNNEDPWT VL4 RASESVDSYGNSFLH LTSNLES QNNEDPWT VH1 TYAMS SISSGGITFYPDSVKG GGDYGNFWFAY VH2 TYAMS SISSGGITFYPDSVKG GGDYGNFWFAY VH3 TYAMS SISSGGITFYPDSVKG GGDYGNFWFAY According to the IMGT numbering system, the results of the VH and VL sequence analysis of the above humanized p-Tau217 antibody are shown in Table 4.

[0038] Table 4: IMGT analysis results of humanized p-Tau217 antibody VH and VL sequences Variable region CDR1 CDR2 CDR3 VL1 ESVDSYGNSF LTS QNNEDPWT VL2 ESVDSYGNSF LTS QNNEDPWT VL3 ESVDSYGNSF LTS QNNEDPWT VL4 ESVDSYGNSF LTS QNNEDPWT VH1 GFTFSTYA ISSGGIT ARGGDYGNFWFAY VH2 GFTFSTYA ISSGGIT ARGGDYGNFWFAY VH3 GFTFSTYA ISSGGIT ARGGDYGNFWFAY Example 3: Specificity verification of humanized p-Tau217 antibody Step 1: Recombinant expression Recombinant expression of mouse p-Tau217 antibody: After the codons of the heavy chain variable region gene of the wild-type mouse p-Tau217 antibody were optimized, they were inserted into the pcDNA3.4 vector containing the mouse IgG heavy chain constant region through the EcoRI and HindIII restriction sites to construct a vector plasmid expressing the complete heavy chain of the antibody; after the codons of the light chain variable region gene of the wild-type mouse p-Tau217 antibody were optimized, they were inserted into the pcDNA3.4 vector containing the mouse IgG light chain constant region through the EcoRI and HindIII restriction sites to construct a vector plasmid expressing the complete light chain of the antibody; the heavy chain plasmid and the light chain plasmid were co-transfected into Expi293F cells to express the antibody, and then purified using a Protein A affinity chromatography column.

[0039] Recombinant expression of humanized p-Tau217 antibody: After the codons of the heavy chain variable region genes of 12 humanized p-Tau217 antibodies were optimized, they were inserted into the pcDNA3.4 vector containing the human IgG heavy chain constant region through the EcoRI and HindIII restriction sites to construct a vector plasmid expressing the complete heavy chain of the antibody; after the codons of the light chain variable region genes of 12 humanized p-Tau217 antibodies were optimized, they were inserted into the pcDNA3.4 vector containing the human IgG light chain constant region through the EcoRI and HindIII restriction sites to construct a vector plasmid expressing the complete light chain of the antibody; the heavy chain plasmid and the light chain plasmid were co-transfected into Expi293F cells to express the antibody, and then purified using a Protein A affinity chromatography column.

[0040] The gene sequence of the mouse IgG heavy chain constant region is as follows: GCCAAAACGACACCCCCATCTGTCTATCCACT GGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCC CATCGAGAAAACCATCTCTAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA; The gene sequence of the constant region of mouse IgG light chain is as follows: GATGCTGCACCAACTGTATCCATCTTCCCACCATCCAG TGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTA; The gene sequence of the constant region of human IgG light chain is as follows: GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGT GGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAG CTTCAACAGGGGAGAGTGTT; The gene sequence of the mouse p-Tau217 antibody after codon optimization is as follows: VL gene: AACATTTTACTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAG GGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTTTGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTGACATCCAACCTAGAATCTGGGGTCCCTGACAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGACCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC; VH gene: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCC CTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGCCATGTCTTGGGTTCGCCAGATTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGGTGGTATCACGTTCTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCACAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGGAGGGGACTATGGTAACTTCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG; The codon-optimized humanized p-Tau217 antibody gene sequence is as follows: VL1 gene: GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGTCCCTGGGCGAG CGGGCTACAATCAACTGCAGAGCCAGCGAGAGCGTGGACTCCTACGGCAATTCTTTTCTGCACTGGTATCAGCAAAAACCTGGCCAGCCTCCAAAGCTGCTGATCTACCTGACAAGCAACCTGGAATCTGGCGTGCCTGATAGATTCAGCGGATCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGGCCGAGGATGTGGCCGTCTACTACTGTCAGCAGAACAACGAGGACCCCTGGACCTTCGGCGGAGGCACAAAGGTGGAAATCAAG; VL2 gene: GACATCGTGATGACCCAGAGCCCCGATAGCCTGGCCGTGTCCCTCGGCGAG CGGGCTACAATCAACTGCAGAGCCAGCGAGAGCGTGGACTCCTACGGCAATAGCTTTCT GCACTGGTATCAGCAAAAACCTGGCCAGCCTCCAAAGCTGCTGATCTACCTGACCAGCA ACCTGGAATCTGGCGTGCCCGACAGATTCAGCGGATCTGGCTCTAGAACAGACTTCACC CTGACAATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTCTACTACTGTCAGCAGAACA ACGAGGATCCTTGGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG; VL3 gene: AATATCGTGATGACCCAGAGCCCTGATTCTCTGGCCGTGTCCCTGGGCGAGC GGGCTACAATCAACTGCAGAGCCAGCGAGAGCGTGGACAGCTACGGCAACAGCTTTCT GCACTGGTATCAGCAAAAACCTGGCCAGCCTCCAAAGCTGCTGATCTACCTGACCAGCA ACCTGGAATCCGGCGTGCCCGATAGATTCAGCGGCTCTGGATCTAGAACAGACTTCACC CTGACAATCAGCAGCCTCCAGGCCGAGGACGTGGCCGTCTACTACTGTCAGCAGAACA ACGAGGACCCCTGGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG; VL4 gene: AATATCGTGCTGACACAGAGCCCCGACAGCCTCGCTGTGTCCCTGGGCGAG CGGGCCACCATCAACTGCAGAGCCAGCGAGAGCGTGGACTCCTACGGCAACTCTTTTCT GCACTGGTATCAGCAAAAACCTGGCCAGCCTCCAAAGCTGCTGATCTACCTGACCAGCA ACCTGGAATCTGGCGTGCCTGATAGATTCAGCGGCAGCGGCTCTAGAACCGACTTCACC CTGACAATCAGCAGCCTGCAGGCCGAGGATGTGGCCGTCTACTACTGTCAGCAGAACA ACGAGGACCCCTGGACCTTCGGCGGAGGAACAAAGGTGGAAATCAAG; VH1 gene: GAAGTGCAGCTGGTTGAGAGCGGCGGAGGCCTGGTGCAACCTGGAGGATC TCTGAGACTGAGCTGTGCTGCTTCTGGATTTACCTTCAGCACATACGCCATGAGCTGGGT GCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTCGCCAGCATCAGCAGCGGCGGCATC ACATTCTACCCCGACAGCGTGAAGGGCAGATTCACCATCTCTAGAGATAATGCCAAGAA CTCCCTCTATCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCG CCAGAGGCGGCGACTACGGCAACTTCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGT GACCGTGTCCAGC; VH2 gene: GAGGTGCAGCTGGTTGAGAGCGGCGGAGGACTGGTGCAACCTGGAGGCAG CCTCAGACTGTCTTGTGCTGCTTCTGGATTTACATTCAGCACATACGCCATGAGCTGGGT GCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTCGCCTCTATCAGCAGCGGCGGCATC ACCTTCTACCCCGACAGCGTGAAGGGCAGATTCACCATCTCCACCGACAACGCCAAGA ACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAAGATACAGCCGTGTACTACTGC GCCAGAGGCGGCGACTACGGCAATTTCTGGTTCGCCTATTGGGGCCAGGGCACCCTGGT GACCGTGTCCAGC; VH3 gene: GAGGTGCAGCTGGTGGAAAGCGGCGGTGGCCTGGTTCAACCTGGAGGATC TCTGAGACTGAGCTGTGCTGCTTCTGGCTTTACATTCAGCACCTACGCCATGAGCTGGGT CAGACAGGCCCCTGGCAAGCGGCTGGAATGGGTGGCCAGCATCAGCAGCGGCGGCATC ACCTTCTACCCCGACAGCGTGAAAGGCAGATTCACCATCTCTACCGACAACGCCAAGAA CTCCCTCTACCTGCGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCG CCAGAGGCGGAGATTACGGCAATTTCTGGTTCGCCTATTGGGGCCAGGGCACACTGGTGACAGTGTCCAGC.

[0041] Step 2: Antigen coating and blocking The commercially available recombinant p-Tau217 antigen (Shanghai Fangyuan Biaopin Biotechnology Co., Ltd., KSDR003-03) and the commercially available recombinant total-Tau antigen (Shanghai Fangyuan Biaopin Biotechnology Co., Ltd., KSDR001-03) were diluted to 1.5 μg / mL with CB buffer, and coated onto a colorless transparent ELISA plate at 100 μl / well; the ELISA plate was blocked with a blocking solution containing BSA at 4°C overnight.

[0042] Step 3: Indirect method to test antibody specificity After blocking, the ELISA plate was shaken off the liquid in the plate and the residual liquid was patted off for use. The purified recombinant expressed antibody was diluted with PBS to a concentration of 1000ng / mL, 250ng / mL, 62.5ng / mL, 15.625ng / mL, 3.906ng / mL, 0.977ng / mL, 0.244ng / mL, 0.061ng / mL, 0.015ng / mL, and then added to the ELISA plate coated with the antigen, 100μl 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 and reacted at 37℃ for 30min. 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. Add 100μL of colorimetric solution to each well, react at 37℃ for 15min, 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.

[0043] The test results are shown in Table 5. As can be seen from Table 5, among the 12 recombinantly expressed humanized p-Tau217 antibodies, RY2, RY3, RY4, RY6, RY7, RY9, and RY12 have significantly improved affinity for the recombinant p-Tau217 antigen.

[0044] Table 5 Antibody specificity test results In addition, the purified recombinantly expressed antibody was tested on an SPR detection instrument (cytiva, Biacore T200), and the antibody affinity constant was finally determined by analyzing the antigen (commercially available recombinant p-Tau217 antigen, Shanghai Fangyuan Biaopin Biotechnology Co., Ltd., KSDR003-03)-antibody binding constant Ka and dissociation constant Kd. The results showed that the affinity constant K of wild-type mouse p-Tau217 antibody for commercially available recombinant p-Tau217 antigen was D The value reaches 9.26×10 - 9 M, affinity constant K of humanized p-Tau217 antibody RY-9 for commercially available recombinant p-Tau217 antigen D The value reaches 5.15×10 - 10 M.

[0045] Example 3: Application of humanized p-Tau217 antibody in diagnosis of Alzheimer's disease Step 1: HRP labeling of humanized p-Tau217 antibody The purified recombinantly expressed humanized p-Tau217 antibody RY-9 was labeled with HRP enzyme.

[0046] Step 2: Coat Tau protein antibody onto ELISA plate A commercially available total-Tau protein antibody (Shanghai Fangyuan Biotechnology Co., Ltd., KSDM001-05) was diluted to a concentration of 1.5 μg / mL with CB buffer and coated onto a colorless transparent ELISA plate at 100 μl per well. The ELISA plate was then blocked overnight at 4°C with a blocking solution containing BSA.

[0047] Step 3: ELISA kit sensitivity verification The HRP-labeled humanized p-Tau217 antibody RY-9 and the commercial total-Tau protein antibody were combined into an ELISA kit, and the commercially available recombinant p-Tau217 antigen (Shanghai Fangyuan Biaopin Biotechnology Co., Ltd., KSDR003-03) was used to verify its sensitivity. The specific steps were as follows: the commercially available recombinant p-Tau217 antigen was diluted to different concentrations, namely 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 15.625pg / mL, 7.8125pg / mL, 3.9pg / mL, 1.95pg / mL, and 0pg / mL. 100μl of the diluted commercially available recombinant p-Tau217 antigen of each concentration was added to the ELISA plate coated with the total-Tau protein antibody and reacted at 37°C 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. Then add 100μL of HRP-labeled humanized p-Tau217 antibody diluted to a certain concentration to each well, and react at 37℃ for 30min. 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; 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.

[0048] The test results are shown in Table 6 and Figure 1 The experimental results showed that when the humanized p-Tau217 antibody RY-9 was used in combination with the commercially available total-Tau protein antibody, it showed a good linear relationship in the enzyme-linked immunosorbent assay (ELISA) within the concentration range of 15pg / mL to 1000pg / mL, and the correlation coefficient R 2The detection limit of commercially available recombinant p-Tau217 antigen was 0.05 pg / mL.

[0049] Table 6 Sensitivity test results Example 4: Application of humanized p-Tau217 antibody in clinical diagnosis of Alzheimer's disease The simoa single-molecule immunoassay was used to determine the concentration of p-Tau 217 in clinical samples. At the same time, the purified recombinantly expressed humanized p-Tau217 antibody RY-9 was applied to the detection of p-Tau217 in clinical samples according to the ELISA method of Example 3, and the correlation of the two detection methods was compared.

[0050] The results are shown in Table 7. The correlation coefficient of the two sets of data calculated by the CORREL function was 0.999999989, that is, when the humanized p-Tau217 antibody RY-9 was used in combination with an antibody that could be paired with it and applied to the ELISA method, the test results had a strong correlation with the sim oa single molecule immunoassay.

[0051] Table 7 Clinical sample test results Plasma sample name Clinical judgment results simoa method measurement value (pg / mL) ELISA measurement value (pg / mL) 442-Plasma MCI / mild 0.0788 0.317 429-Plasma Dementia 0.8671 4.035 625-Plasma MCI / mild 0.0918 0.379 620-Plasma Dementia 0.8227 3.826 635-Plasma MCI / mild 0.1383 0.598 The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A p-Tau217 antibody, characterized in that: The light chain variable region of the p-Tau217 antibody comprises: CDRL1 amino acid sequence: RASESVDSYGNSFLH, CDRL2 amino acid sequence: LTSNLES, CDRL3 amino acid sequence: QQNNEDPWT; the heavy chain variable region of the p-Tau217 antibody comprises: CDRH1 amino acid sequence: TYAMS, CDRH2 amino acid sequence: SISSGGITFYPDSVKG, CDRH3 amino acid sequence: GGDYGNFWFAY.

2. The p-Tau217 antibody according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of the p-Tau217 antibody is: NILLTQSPASLAVSLGQRATISCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPWTFGGGTKLEIK; the amino acid sequence of the heavy chain variable region of the p-Tau217 antibody is: EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQIPEKRLEWVASISSGGITFYPDSVKGRFTISTDNARNILYLQMSSLRSEDTAMYYCARGGDYGNFWFAYWGQGTLVTVSA.

3. A method for humanizing the p-Tau217 antibody according to claim 2, characterized in that: The complementarity determining regions of the p-Tau217 antibody were grafted onto a human framework, and then the amino acids in the light chain framework region and / or the heavy chain framework region were backmutated to restore the affinity of the humanized antibody.

4. The method according to claim 3, characterized in that: The back mutation of the light chain framework region is selected from any one or more of G72R, D1N, and M4L; the back mutation of the heavy chain framework region is selected from any one or more of R71T and G44R.

5. A humanized p-Tau217 antibody, characterized in that: Obtained by transformation of the method described in claim 4.

6. The humanized p-Tau217 antibody according to claim 5, characterized in that: The amino acid sequence of the light chain variable region of the humanized p-Tau217 antibody is selected from NIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK, DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK SSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK, DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTDFT LTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK,NIVLTQSPDSLAVSLGERATINCRASESVDSYGNSFLHWYQQKPGQPPKLLIYLTSNLESGVPDRFSGSGSRTD FTLTISSLQAEDVAVYYCQQNNEDPWTFGGGTKVEIK; the heavy chain variable region amino acid sequence of the humanized p-Tau217 antibody is selected from EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKRLEWVASISSGGITFYPDSVKGRFTISTDNAKNSLYLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS, EVQLVESGGGLVQPGGSLRLSCAASGF TFSTYAMSWVRQAPGKGLEWVASISSGGITFYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS,EVQLVESGGGLVQPGG Any of SLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVASISSGGITFYPDSVKGRFTISTDNAKNSLYLQMNSLRAEDTAVYYCARGGDYGNFWFAYWGQGTLVTVSS.

7. An isolated nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the humanized p-Tau217 antibody according to any one of claims 5 to 6.

8. A method for preparing a humanized p-Tau217 antibody, characterized in that: The nucleic acid molecule described in claim 7 is cloned into a pcDNA3.4 expression vector containing a human IgG constant region to obtain a recombinant expression vector; the recombinant expression vector is transferred into Expi293F cells to obtain recombinant cells; the recombinant cells are cultured, and the humanized p-Tau217 antibody is isolated and purified from the culture.

9. Use of the p-Tau217 antibody according to any one of claims 1 to 2 or the humanized p-Tau217 antibody according to any one of claims 5 to 6 in the preparation of a kit for assisting the diagnosis of Alzheimer's disease.

10. Use of the p-Tau217 antibody according to any one of claims 1 to 2 or the humanized p-Tau217 antibody according to any one of claims 5 to 6 in detecting p-Tau217 protein, characterized in that: The applications described do not include applications in disease diagnosis and treatment.

Images