Anti-thyrotropin receptor monoclonal antibody 1A4 and use thereof

Abstract

The application relates to the field of biotechnology, in particular to an anti-thyrotropin receptor monoclonal antibody 1A4 and application. The application discloses an anti-thyrotropin receptor monoclonal antibody 1A4, the sequence of the monoclonal antibody 1A4 comprises a heavy chain variable region shown in SEQ ID NO. 8 and a light chain variable region shown in SEQ ID NO. 17; the monoclonal antibody 1A4 can have high affinity with TSHR289 protein, can bind to natural wild type TSHR, has thyroid stimulating activity, can be inhibited by TRAb in GD patient serum to compete with the binding to TSHR, and has other biological activities, and can be used for the preparation or development of an in vitro diagnostic kit of specific therapeutic drugs against stimulating TRAb, provides a theoretical basis and a powerful tool for the detection of TRAb types in clinic, and can be used for the development of specific therapeutic drugs against stimulating TRAb antibodies.

Description

[0001] The present application claims priority from the prior application with the application date of September 27, 2024, the application number of CN2024113608076, and the invention name of "Anti-Thyrotropin Receptor Monoclonal Antibody 1A4 and Application", the entire contents of the prior application are embodied in the present application. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to an anti-thyrotropin receptor monoclonal antibody 1A4 and application. BACKGROUND

[0003] Graves disease (GD) is an autoimmune thyroid disease caused by the interaction of natural, environmental and genetic factors. Thyrotropin receptor antibody (TRAb) is the core mechanism of GD pathogenesis, which is mainly an autoantibody against the thyrotropin receptor (TSHR).

[0004] TRAb not only plays a core role in the pathogenesis of GD, but is also closely related to the outcome and recurrence of the disease. Accurate detection of TRAb levels is a key indicator for predicting the course of GD at the initial diagnosis and treatment stages, guiding treatment, and preventing recurrence. With the rapid development of biological therapy, hyperthyroidism caused by autoimmune thyroid disease has attracted much attention, and research on TRAb-related antisense peptides and monoclonal antibodies has become a hot topic in the field.

[0005] TRAb is an oligoclonal IgG produced in GD patients, first discovered in GD hyperthyroidism patients in 1956, and initially referred to as long-acting thyroid stimulator. TRAb is a polyclonal antibody, mainly divided into stimulating antibody (TSAb), blocking antibody (TBAb), and neutral antibody. Among them, TSAb binds to TSHR to initiate the second signal, playing a major decisive role in the pathogenesis of GD hyperthyroidism. TBAb is believed to play a major role in the remission period of the disease, and may be the cause of GD patients' concurrent hypothyroidism in the later stage. Neutral antibody binding to TSHR may induce apoptosis and oxidative stress in the thyroid gland. Currently, although biological analysis and bridge technology have been developed to analyze and detect TRAb, the traditional competitive assay is still used in clinical detection of TRAb. However, this competition can only determine the presence of TRAb in GD patient serum, and cannot completely distinguish TSAb, TBAb, and neutral antibody. The other two detection methods have not been widely used in clinical detection due to long cycle and high detection components. This results in clinicians being unable to accurately determine whether GD patients are in the active or remission stage, and unable to achieve individualized precision treatment for GD patients, which may lead to delays and exacerbations of the patient's condition.

[0006] Currently, the clinical treatment of Graves' disease (GD) still relies on three methods that have been used for decades: antithyroid drugs (ATDs). 131 IgI therapy and thyroidectomy. These treatments can only improve thyroid function in some patients in the short term. In most patients, the autoimmune system quickly reactivates, and serum TRAb levels rebound or even rise higher than before. 131 Patients treated with thyroid hormone replacement therapy (TIA) may experience a short-term surge in serum TRAab levels, potentially triggering Graves' disease (GD) and accelerating its progression. The root cause is that these treatments do not fundamentally address the issue of abnormal TRAab production in GD patients; they only provide short-term relief by suppressing thyroid hormone production, or may lead to a remission phase of the GD disease itself. 131 I-treatment may have damaged the body's immune system, leading to excessive suppression of T-cell activation, reduced immune cell activity, and uncontrolled antibody release. Meanwhile, emerging targeted therapies such as lymphocyte-targeting biologics (rituximab, RTX), small molecule TSHR antagonists (ANTAG-3, VA-K-14, and S37a), TBA active targeted drugs (K1-70), and TSHR mixed peptides (ATX-GD-59) have also been shown to have limited efficacy in GD patients.

[0007] Given the crucial role of TRAbs in the development of Graves' disease (GD) and related conditions, treatments targeting TRAbs are receiving increasing attention. Pathological stimulant TRAbs (TSAbs) competitively bind to TSHR with their natural ligand TSH, initiating a signaling pathway to produce cyclic adenosine monophosphate (cAMP), which stimulates thyroid hormone synthesis and secretion—a major pathogenic mechanism in GD hyperthyroidism. TSHR monoclonal antibodies (MAbs), competitive antagonists of TSAbs, have long been a focus of attention as potential therapeutic agents for GD; therefore, all the above research has concentrated on TSHR and TRAbs.

[0008] TSHR belongs to the G protein-coupled receptor superfamily, which is present on the plasma membrane of thyroid follicular epithelial cells, has a full length of 764 amino acid residues, and includes a large extracellular domain (ECD), a seven-helix transmembrane domain (TMD), and a C-terminal tail. The ECD contains a TSHR leucine-rich domain (LRD) and a hinge region, binds TSAb, and activates G protein through conformational changes of the TMD. The ECD contains amino acid residues 22-410 after removing a signal peptide of the first 21 amino acid residues, and has 6 glycosylation sites. After transcription and translation of TSHR, the TSHR is cleaved into an A subunit and a B subunit linked by 2 disulfide bonds. The A subunit is a secreted soluble hyperglycosylated protein containing TSHR amino acid residues 22-289, is located in the ECD, and includes all glycosylation sites. The TSHR A subunit mainly mediates the maturation of TRAb with high affinity to TSHR in GD patients, and it is found through immunization of mice with an adenovirus encoding a human TSHR gene that the shed A subunit can induce hyperthyroidism in most mice, and the increase of TRAb can be detected in the serum of the mice. The shed A subunit can be released to thyroid lymphocytes and captured by antigen-presenting cells. The mannose receptors expressed on these cells bind to the highly glycosylated A subunit, are processed into polypeptides, and are presented to T cells, which can in turn lead to TSAb response. Therefore, the TSHR A subunit is currently considered as the main immunogen of GD.

[0009] The preparation of monoclonal antibodies using the TSHR A subunit as the main immunogen can help to explore the structure and function of TSAb. Although animal-derived monoclonal antibodies prepared by immunizing mice and hamsters with different TSHR antigen fragments and human-derived monoclonal antibodies directly isolated from human peripheral blood have been obtained by different laboratories, these do not change the difficult situation of diagnosis and treatment of GD in clinic. GD is still a refractory disease so far. The present application attempts to screen and use antigen fragments of the main pathogenic sites of TRAb to prepare TSHR MAbs with certain activity, to establish specific detection and treatment means of corresponding autoantibodies using the TSHR MAbs as targets, to play an active role in predicting the disease course, guiding treatment, and preventing recurrence of GD, and to have far-reaching significance in exploring the pathological mechanism of GD. SUMMARY

[0010] In order to solve the above technical problems, the present application aims to provide an anti-thyroid stimulating hormone receptor monoclonal antibody 1A4 and application. Specifically includes the following contents:

[0011] In a first aspect, the present application provides an anti-thyroid stimulating hormone receptor monoclonal antibody 1A4, wherein the monoclonal antibody 1A4 comprises an antibody heavy chain and an antibody light chain.

[0012] The variable region CDR of the antibody heavy chain comprises CDR1 with an amino acid sequence as shown in SEQ ID NO. 1, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 3.

[0013] The variable region CDR of the antibody light chain comprises CDR1 with an amino acid sequence as shown in SEQ ID NO. 10, CDR2 with an amino acid sequence as shown in SEQ ID NO. 11, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 12.

[0014] Preferably, the variable region FR of the antibody heavy chain comprises FR1 with an amino acid sequence as shown in SEQ ID NO. 4, FR2 with an amino acid sequence as shown in SEQ ID NO. 5, FR3 with an amino acid sequence as shown in SEQ ID NO. 6, and FR4 with an amino acid sequence as shown in SEQ ID NO. 7.

[0015] The variable region FR of the antibody light chain comprises FR1 with an amino acid sequence as shown in SEQ ID NO. 13, FR2 with an amino acid sequence as shown in SEQ ID NO. 14, FR3 with an amino acid sequence as shown in SEQ ID NO. 15, and FR4 with an amino acid sequence as shown in SEQ ID NO. 16.

[0016] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is as shown in SEQ ID NO. 8, and the amino acid sequence of the variable region of the antibody light chain is as shown in SEQ ID NO. 17.

[0017] In a second aspect, the present application provides a nucleic acid encoding the antibody heavy chain and the antibody light chain of the monoclonal antibody 1A4 according to the first aspect.

[0018] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is as shown in SEQ ID NO. 9, and the nucleotide sequence encoding the variable region of the antibody light chain is as shown in SEQ ID NO. 18.

[0019] In a third aspect, the present application provides use of the monoclonal antibody 1A4 according to the first aspect in the preparation of a specific therapeutic drug for resisting stimulating TRAb.

[0020] Preferably, the specific therapeutic drug is an immunosuppressant.

[0021] In a fourth aspect, the present application provides use of the monoclonal antibody 1A4 according to the first aspect in the preparation of a reagent or kit for detecting TRAb.

[0022] In a fifth aspect, the present application provides use of the monoclonal antibody 1A4 of the first aspect above in the preparation of a diagnostic or prognostic kit for a TSHR-related disease.

[0023] Preferably, the TSHR-related disease is an autoimmune thyroid disease.

[0024] Preferably, the autoimmune thyroid disease comprises Graves' disease.

[0025] The beneficial effects of the present application are: ① the present application first prepared a recombinant TSHR289 protein, immunized mice with adenovirus-encoded TSHR A subunit, and purified the recombinant TSHR289 protein as a screening protein, thereby obtaining an anti-thyroid stimulating hormone receptor monoclonal antibody 1A4, which comprises a heavy chain variable region as shown in SEQ ID NO. 8 and a light chain variable region as shown in SEQ ID NO. 17; ② the monoclonal antibody 1A4 has high affinity with the recombinant TSHR289 protein, and can also bind to the natural wild-type TSHR; ③ the binding of the monoclonal antibody 1A4 to the TSHR receptor can be competitively inhibited by TRAb in GD patient serum, and has the biological characteristics of human TRAb, thereby providing a theoretical basis and powerful tool for the detection of TRAb in clinic; ④ the monoclonal antibody 1A4 has high thyroid stimulating activity in vitro and in vivo, and can be used as an anti-TSHR monoclonal antibody passive immunization preparation product or developed into an in vitro diagnostic kit. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Purity analysis results of the monoclonal antibody 1A4; wherein M is a protein marker, lane 1 is the reduced purified anti-TSHR monoclonal antibody 1A4-IgG, and lane 2 is the non-reduced purified anti-TSHR monoclonal antibody 1A4-IgG;

[0027] Figure 2 Affinity detection results of the monoclonal antibody 1A4;

[0028] Figure 3 Binding force analysis results of the monoclonal antibody 1A4 and the wild-type TSHR;

[0029] Figure 4 Activity analysis results of the monoclonal antibody 1A4;

[0030] Figure 5The TRAb in the serum of GD patients is detected by competition ELISA, which competes to inhibit the binding of HRP-labeled monoclonal antibody 1A4 to TSHR; wherein, A: the dilution ratio of 1A4 (after dilution at 2000 times, 4000 times, and 8000 times), the binding ability of 1A4 to TSHR is detected by ELISA to determine the optimal dilution ratio of 1A4; B: 1A4 is diluted at the optimal dilution ratio, the binding of 1A4 to TSHR in the serum of GD patients is detected by ELISA, and the serum of 14 normal healthy patients is used as a control;

[0031] Figure 6 The results of the activity detection of monoclonal antibody 1A4 in vivo; wherein, A is the serum TT4 detection of mice in the control group and the monoclonal antibody 1A4 injection group; B is the thyroid HE section of the mice in the control group, I is under 100x microscope, and II is under 400x microscope; C is the thyroid HE section of the mice in the monoclonal antibody 1A4 injection group, all under 400x microscope, the red arrow is the proliferation of follicular epithelial cells, and the black arrow is the expansion of follicles. DETAILED DESCRIPTION

[0032] The embodiments of the present application are described in detail below, and it should be noted that the embodiments described below are exemplary and are used to explain the present application, and should not be understood as a limitation of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements fall within the protection scope of the present application.

[0033] Sf9 cells and High Five cells used in the following examples were from Thermo Fisher Scientific; SP2 / 0 mouse myeloma cells were from the Fourth Research Institute of Lanzhou Institute of Biological Products Co., Ltd.; the first 289 amino acid residues of human TSHR were used as a template to amplify the TSHR289 construct, which was cloned into pFastBac1 and constructed by Shanghai Generay Biotech Co., Ltd.; the adenovirus expressing human TSHR A subunit was constructed by Shanghai Jikai Co., Ltd.; sequencing was completed by Shanghai Generay Biotech Co., Ltd.; the plasmid extraction kit was purchased from Qiagen; Lip2000 was purchased from Thermo Fisher Scientific; PEG1450 was purchased from Beijing Boao Sun Bioengineering Co., Ltd.; Sf-900™ II SFM medium, 1640 medium, F12K medium, fetal bovine serum, HT and HAT were purchased from Thermo Fisher Scientific; TSHR antibody was purchased from Santa Cruz Biotechnology; Rabbit anti-mouse IgG-(HRP) was purchased from Merck; SIM HF Expression Medium was purchased from Yiqiao Shen Zhou; affinity purification medium was purchased from GE; a fluorescence microscope was purchased from ZEISS; an ELISA multi-well plate reader was purchased from Molecular Devices; an ELISA plate washer was purchased from BIO-RAD; a flow instrument was purchased from BD; and a cAMP detection kit was purchased from R&D.

[0034] Example 1 Preparation of monoclonal antibody 1A4

[0035] 1. Expression of recombinant TSHR289 protein

[0036] The recombinant pFastBac1 containing TSHR289 gene (expressing the first 289 amino acid residues of human TSHR) was constructed by Shanghai Generay Biotech Co., Ltd. TM 1. The plasmid TOP10 E. coli was inoculated into LB medium and cultured overnight. The target plasmid was extracted and transformed into DH10 Bac E. coli, inoculated into SOC medium and cultured, then inoculated into a selection plate and cultured overnight. A well-grown white single colony was selected, inoculated into SOC medium and cultured overnight, and then sent to Shanghai Generay Biotech Co., Ltd. for sequencing. The correct one was expanded and the plasmid was extracted. The recombinant Bacmid DNA was transfected into well-grown sf9 cells using Lip2000, and primary virus was obtained after one week. The cell supernatant was collected, centrifuged at 1000g for 5 min, and filtered through a 0.22um filter to obtain the virus. The primary virus was used to infect sf9 cells to expand the virus amount. The virus was inoculated into High Five TMCells, the best time of protein harvest was obtained 72h after infection. Western blot was used to detect the expression of TSHR289. Recombinant protein TSHR289 was obtained by affinity chromatography with NI column. SDS-Page was used to detect the purity of recombinant protein TSHR289.

[0037] 2. Mouse immunization

[0038] 6-8 weeks old female BALB / c mice were immunized with adenovirus expressing human TSHR A subunit by intramuscular injection. Immunization was performed once a week for 3 weeks. One week after the second and third immunization, blood samples were collected from the tail vein to detect the serum titer. The mice with higher titer were immunized for the third time and the booster immunization, respectively.

[0039] 3. Serum titer determination

[0040] Blood samples were collected from the tail vein at different time points. The blood was incubated at 37℃ for 1 hour and at 4℃ for 2 hours. Then, the serum was obtained by centrifugation at 4000 rpm for 30 minutes. The serum was diluted by 1:500 to 1:160000. The purified TSHR289 recombinant protein was diluted with PBS to 5ug / mL, and 100μL / well was coated on the enzyme-labeled plate for overnight blocking. 100μL / well of the diluted mouse serum (incubated at 37℃ for 1 hour) was added. The diluent was discarded, and the plate was washed with PBST for 5 times. HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody was added (incubated at 37℃ for 1 hour). The diluent was discarded, and the plate was washed with PBST for 5 times. A and B mixed solution was added, and incubated at 37℃ for 15 minutes. Then, 50μL of stop solution was added to each well, and the OD 450 value was read by enzyme-labeled plate reader.

[0041] 4. Preparation of hybridoma

[0042] Before fusion, the peritoneal macrophages of 6-8 weeks old BALB / c female mice were prepared as feeder cells. The spleen cells of the mouse with the highest antibody titer were selected and fused with SP2 / 0 myeloma cells in a 10:1 ratio. PEG1450 was added for fusion, and after fusion, HAT selection medium containing 20% FBS was added to gently resuspend the fusion cells. Then, 150μL per well was added to the 96-well cell culture plate coated with feeder cells. The plate was incubated in a 37℃, 5% CO2 cell incubator. The next day, the growth of the cells was observed. Two days after fusion, half of the HAT selection medium containing 20% FBS was replaced. Four days after fusion, the cell fusion was observed under a microscope, and the culture wells with hybridoma cell clones were labeled.

[0043] 5. Screening and cloning of positive hybridoma cells

[0044] The hybridoma cell culture supernatant was subjected to whole plate ELISA detection, the first round of screening used indirect ELISA to screen positive hybridoma cell clones, the primary antibody was hybridoma cell culture supernatant, and the secondary antibody was HRP-labeled anti-mouse antibody. Try to select positive hybridoma cells with high absorbance OD 450 The positive hybridoma cells with high reading value, small number of cell clones and good cell state were subjected to cloning culture. Limited dilution method was used for 3-5 rounds of screening. In each round of cloning, the positive single clone hybridoma cells were expanded and passaged, and indirect ELISA detection was also performed during the passage to detect the passage stability of the cells, and the cell cryopreservation was also done.

[0045] 6. Large-scale preparation and purification of monoclonal antibodies

[0046] The positive single clone hybridoma cells were transferred into a 24-well cell culture plate for culture, and then transferred into a 6-well cell culture plate, a T25 and a T75 cell culture flask for expansion culture. When the cells in the cell culture flask rapidly proliferated, the culture cells were collected, centrifuged at 1000 rpm for 5 min, 1×10 6 The cells were injected into the abdominal cavity of BALB / c female mice which were sensitized in advance. After one week, the mouse ascites was collected, centrifuged at 12000 rpm for 30 min, resuspended with PBS and filtered with a 0.45um filter membrane. The mouse hybridoma antibody was purified by Protein G pre-packed column. The protein concentration of the monoclonal antibody was determined by GE NanoVue ultramicro spectrophotometer Protein A 280 method. The bacteria were filtered and stored at 2-8℃. The anti-TSHR monoclonal antibody obtained by screening is numbered 1A4; the sequencing results show that the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are:

[0047] CDR1: GFSLTGYG (as shown in SEQ ID NO. 1);

[0048] CDR2: IWGDGST (as shown in SEQ ID NO. 2);

[0049] CDR3: AREREDGYYDAMDY (as shown in SEQ ID NO. 3);

[0050] The amino acid sequences of the four FR regions of the heavy chain variable region are:

[0051] FR1: EVQLEESGPGLVAPSQSLSITCTVS (as shown in SEQ ID NO. 4);

[0052] FR2: VNWVRQPPGKGLEWLGM (as shown in SEQ ID NO. 5);

[0053] FR3: DYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYC (as set forth in SEQ ID NO. 6); FR4: WGQGTSVTVSS (as set forth in SEQ ID NO. 7);

[0054] amino acid sequence of the heavy chain variable region: EVQLEESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPP GKGLEWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARERED GYYDAMDYWGQGTSVTVSS (as set forth in SEQ ID NO. 8);

[0055] nucleotide sequence of the heavy chain variable region: GAGGTGCAGCTGGAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGGGTGATGGAAGCACAGACTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGAGAGAGGATGGTTACTACGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (as set forth in SEQ ID NO. 9).

[0056] The amino acid sequences of CDR1, CDR2, CDR3 of the light chain variable region are:

[0057] CDR1: QSLLYSSNQKNY (as set forth in SEQ ID NO. 10);

[0058] CDR2: WAS (as set forth in SEQ ID NO. 11);

[0059] CDR3: QQYYSYPR (as set forth in SEQ ID NO. 12);

[0060] The amino acid sequences of the four FR regions of the light chain variable region are:

[0061] FR1 : DIVMTQSPSSLAVSVGEKVTMSCKSS (shown in SEQ ID NO. 13);

[0062] FR2: LAWFQQNPGQSPKLLIY (shown in SEQ ID NO. 14);

[0063] FR3: TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC (shown in SEQ ID NO. 15);

[0064] FR4: GRSVEAPSWKSN (shown in SEQ ID NO. 16);

[0065] Amino acid sequence of the light chain variable region: DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLA WFQQNPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYP RGRSVEAPSWKSN (shown in SEQ ID NO. 17);

[0066] Nucleotide sequence of the light chain variable region: GACATTGTGATGACCCAGTCTCCATCCTCCCTAGCTGTGT CAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTTCCAGCAGAATCCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCACGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (shown in SEQ ID NO. 18).

[0067] Example 2: Detection of properties of monoclonal antibody 1A4

[0068] 1. Analysis of purity of the antibody

[0069] The present example adopts cell culture method and mouse ascites method to prepare a large amount of anti-TSHR monoclonal antibody 1A4, and the cell culture supernatant and the ascites after crude extraction are subjected to Protein G. After sample elution, the protein concentration is measured by NanoVue ultramicro spectrophotometer to be 430 μg / ml. Because the molecular weight of the monoclonal antibody is large (mostly 150-180 kDa), the monoclonal antibody is subjected to β-mercaptoethanol reduction treatment, and the purity of the purified monoclonal antibody is analyzed by reducing and non-reducing SDS-PAGE electrophoresis.

[0070] The results are shown in Figure 1 The figure shows that the monoclonal antibody is reduced by the β-mercaptoethanol reducing agent to 50 kDa (heavy chain) and 25 kDa (light chain), and the molecular weight of the non-reduced monoclonal antibody is about 150 kDa, which is consistent with the size of IgG molecular weight. The electrophoresis results show that the Protein G medium affinity purification method is used to obtain high-purity monoclonal antibody.

[0071] 2. Affinity detection of the antibody

[0072] The present example uses surface plasmon resonance analysis to detect the affinity and kinetic properties of anti-TSHR monoclonal antibody 1A4 and TSHR289 fusion protein. 10 mM sodium acetate solution with pH = 4.5 is used as the coating liquid, and CM5 chip is activated under the action of EDC / NHS, ethanolamine is used for blocking, the coating flow rate is 10 μL / min, the running buffer is HBS-EP buffer, and the purified TSHR289 fusion protein is coated on the CM5 chip. The monoclonal antibody 1A4 is diluted by HBS-EP Buffer to 3.0 μg / mL, and is flowed through the CM5 chip coated with purified TSHR289 in turn, and the injection flow rate is 30 μL / min. After the binding is completed, it is regenerated in Gly-HCL buffer with pH 1.5. After the reaction is completed, Biacore T200 Evaluation Software 1.0 is used for analysis.

[0073] The results are shown in Figure 2 The affinity assay shows that the affinity of the purified TSHR289 protein and the monoclonal antibody 1A4 is 6.333 x 10 10 .

[0074] 3. Analysis of the binding force of the antibody and the wild type TSHR

[0075] The present embodiment uses flow cytometry to detect the binding ability of anti-TSHR monoclonal antibody 1A4 to wild-type human TSHR stably transfected into CHO cells. After trypsin digestion of well-grown CHO cells stably transfected with wild-type human TSHR, the cells were counted, centrifuged at 1000 rpm for 5 min, and resuspended in 2% BSA. After adding Fcblock, the Fab fragment binding test of monoclonal antibody 1A4 was performed under the same conditions. 1x10 6 cells / tube were taken from positive cells and untransfected cells, respectively, and 10 μg of purified monoclonal antibody 1A4 was added. After incubation at room temperature in the dark for 60 min, a control group was set up by adding the same volume of PBS. After washing with 2% BSA for 3 times, Per-CP labeled secondary antibody was added and incubated at room temperature in the dark for 45 min. After washing with 2% BSA for 3 times, resuspension was performed, and detection and analysis were performed using a BD AccuriC6 flow cytometer. The results were analyzed using flowjo software.

[0076] The results are shown in Figure 3 Table 1. The binding rates of IgG and Fab fragments of anti-TSHR monoclonal antibody 1A4 to wild-type TSHR were 76.3% and 82.3%, respectively.

[0077] 4. Activity analysis of antibody

[0078] The present embodiment uses competitive ELISA technology to detect the activity of anti-TSHR monoclonal antibody 1A4 in stimulating the production of cAMP by CHO cells stably transfected with wild-type human TSHR. After trypsin digestion of well-grown CHO cells stably transfected with wild-type human TSHR, the cells were counted and 5x10 4 cells / well were evenly plated in a 96-well cell culture plate. After 24 h of culture in F12K complete medium, the cell adhesion was observed. After the cells adhered well, the original culture medium was discarded, and the cells were cultured in serum-free medium for 2 h. Then, 1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine were added to each well, and the purified monoclonal antibody 1A4 was diluted to different concentrations. The mixture was incubated at 37°C for 4 h. After washing with PBS for 3 times, 150 μL / well of cell lysis solution was added to the 96-well plate, and the plate was repeatedly frozen and thawed 3 times at -80°C. Finally, the plate was incubated overnight at -80°C. The next day, the detection was performed according to the manufacturer's method. After the experiment was repeated 3 times, the average value was taken for statistical analysis.

[0079] The results are shown in Figure 4 Table 2. The EC 50 of IgG and Fab fragments of anti-TSHR monoclonal antibody 1A4 were 0.58 μg / ml and 0.12 μg / ml, respectively.

[0080] 5. Analysis of antibody competition with GD patient serum TRAb for binding to receptor

[0081] This example uses a competitive ELISA technique to detect TRAb in GD patient serum that competes to inhibit the binding of TSHR by monoclonal antibody 1A4 to TSHR. 46 human serum samples were collected, including 32 serum samples from GD patients with positive TRAb and 14 serum samples from normal healthy people (without any other diseases including any autoimmune diseases) with negative TRAb. The serum samples were mixed with diluted HRP-labeled 1A4 monoclonal antibody and added to an ELISA plate coated with TSHR antigen in a commercial kit. After incubation at 37°C for 60 min, the plate was washed 5 times with PBST, mixed A+B solution was added, and after incubation at 37°C for 15 min, color developing solution C was added. The microplate was shaken and read at OD 450 nm. The inhibition rate calculation formula is: Inhibition rate (%) = (B0-Bi) / B0x100%. Bi is the OD value of the experimental sample, and B0is the OD value of the control sample.

[0082] The results are shown in Figure 5 A: 1A4 dilution ratio (2000-fold, 4000-fold, and 8000-fold dilution), ELISA detection of the ability to compete 1A4 with TSHR, determination of the optimal dilution of 1A4; B: 1A4 was diluted at a ratio of 1:4000, and ELISA was used to detect the competition of GD patient serum TRAb to inhibit the binding of 1A4 to TSHR. The results show that the binding of the monoclonal antibody 1A4 to TSHR can be competitively inhibited by most of the GD patient serum TRAb (with 50% inhibition rate as the standard for inhibition).

[0083] 6. Detection of the activity of the antibody in vivo

[0084] This example uses ELISA technology and HE sections to detect the biological activity of anti-TSHR monoclonal antibody 1A4 in animals. Purified monoclonal antibody 1A4 was injected intraperitoneally into 6-8 week old BALB / c female mice at a dose of 250 μg, twice a week for 3 weeks. The control group was injected with PBS in the same way. Two days after the last injection, the mice were anesthetized with an overdose of isoflurane, blood was collected, and the mouse thyroid tissue was fixed, dehydrated, and embedded for HE staining.

[0085] The results are shown in Figure 6 The serum TT4 level of the 1A4 injection group of mice was (7.1±0.7 ng / ml) ng / ml (MEAN±SEM; n=10), which was not statistically significant compared with the control mice (5.9±0.6 ng / ml) (A shown in Figure 6 On the HE sections, it was found that the mouse thyroid follicular epithelial cells showed mild hyperplasia, and the follicle shape and size were not uniform, with follicular expansion (B shown in Figure 6B and C as shown in the figure.

[0086] From the above activity analysis results can be known:

[0087] The present application successfully prepared the anti-TSHR289 protein monoclonal antibody 1A4, which not only can have high affinity with purified TSHR289 protein, but also can combine with natural wild type TSHR. Secondly, the monoclonal antibody 1A4 also has thyroid stimulating activity, that is, even at a lower concentration (μg / ml level) can stimulate CHO cells transfected with human TSHR full-length receptor to produce cAMP. The thyroid stimulating activity of the monoclonal antibody 1A4 described in the present application is similar to the activity of the TRAb in the human serum currently reported. At the same time, the present application also detects that the Fab segment of the monoclonal antibody 1A4 also shows similar activity.

[0088] The present application uses HRP labeled monoclonal antibody 1A4, and the competition ELISA detection finds that the combination of the monoclonal antibody 1A4 and TSHR can be competitively inhibited by TRAb in the GD patient serum. It is shown that the monoclonal antibody 1A4 has the biological activity of human TRAb, and provides a theoretical basis and a powerful tool for the current clinical TRAb detection.

[0089] After injecting the female BALB / c female mice with the purified monoclonal antibody 1A4 of the present application, compared with the PBS injection group, the serum TT4 level does not appear obvious change. But the follicular epithelial cells of the thyroid of the 1A4 injected mice are found to have slight hyperplasia, the follicle size is not uniform, and the follicle appears typical expansion pathological changes. It is shown that although the typical hyperthyroidism performance of the mice is not induced, the monoclonal antibody 1A4 still exhibits certain thyroid stimulating activity in vivo.

[0090] In summary, the monoclonal antibody 1A4 described in the present application has higher thyroid stimulating activity in vitro and in vivo, the combination of which with TSHR can be competitively inhibited by TRAb in the GD hyperthyroidism patient serum. And compared with the mouse-derived anti-TSHR289 monoclonal antibody currently reported, ① the monoclonal antibody 1A4 described in the present application has high affinity with natural wild TSHR; ② has strong thyroid stimulating activity; ③ the combination of which with TSHR can be competitively inhibited by TRAb in the GD hyperthyroidism patient serum. It is possible to be used as an anti-TSHR monoclonal antibody passive immunization preparation product or to develop an in vitro diagnostic kit.

[0091] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An anti-thyrotropin receptor monoclonal antibody 1A4, characterized in that, The monoclonal antibody 1A4 comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain comprises CDR1 with an amino acid sequence as shown in SEQ ID NO. 1, CDR2 with an amino acid sequence as shown in SEQ ID NO. 2, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 3; The variable region CDR of the antibody light chain comprises CDR1 with an amino acid sequence as shown in SEQ ID NO. 10, CDR2 with an amino acid sequence as shown in SEQ ID NO. 11, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

12.

2. The monoclonal antibody 1A4 of claim 1, wherein The variable region FR of the antibody heavy chain comprises FR1 with an amino acid sequence as shown in SEQ ID NO. 4, FR2 with an amino acid sequence as shown in SEQ ID NO. 5, FR3 with an amino acid sequence as shown in SEQ ID NO. 6, and FR4 with an amino acid sequence as shown in SEQ ID NO. 7; The variable region FR of the antibody light chain comprises FR1 with an amino acid sequence as shown in SEQ ID NO. 13, FR2 with an amino acid sequence as shown in SEQ ID NO. 14, FR3 with an amino acid sequence as shown in SEQ ID NO. 15, and FR4 with an amino acid sequence as shown in SEQ ID NO.

16.

3. The monoclonal antibody 1A4 of claim 1, wherein, The amino acid sequence of the variable region of the antibody heavy chain is as shown in SEQ ID NO. 8, and the amino acid sequence of the variable region of the antibody light chain is as shown in SEQ ID NO.

17.

4. A nucleic acid, characterized in that, The nucleic acid encodes the antibody heavy chain and the antibody light chain of the monoclonal antibody 1A4 of any one of claims 1-3.

5. The nucleic acid of claim 4, wherein, The nucleotide sequence encoding the variable region of the antibody heavy chain is as shown in SEQ ID NO. 9, and the nucleotide sequence encoding the variable region of the antibody light chain is as shown in SEQ ID NO.

18.

6. Use of the monoclonal antibody 1A4 of any one of claims 1-3 in the preparation of a reagent or kit for detecting TRAb.

7. Use of the monoclonal antibody 1A4 of any one of claims 1-3 in the preparation of a diagnostic or predictive kit for a disease related to TSHR.

8. Use according to claim 7, wherein the compound is ###0002### The disease related to TSHR is an autoimmune thyroid disease.

9. Use according to claim 8, wherein the compound is ###0002### The autoimmune thyroid disease comprises Graves' disease.

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