Monoclonal antibody 1A4 for resisting thyroid stimulating hormone receptor and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-thyroid stimulating hormone receptor monoclonal antibody 1A4 and application thereof. The invention discloses a monoclonal antibody 1A4 for resisting a thyroid stimulating hormone receptor. The sequence of the monoclonal antibody 1A4 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; the monoclonal antibody 1A4 has high affinity with TSHR289 protein, can be combined with natural wild type TSHR, has thyroid irritating activity, can competitively inhibit combination of TRAb and TSHR in serum of GD patients, has strong biological activity, can be used for preparation of specific therapeutic drugs for resisting irritating TRAb or development of in vitro diagnostic kits, and has broad application prospects. A theoretical basis and a powerful tool are provided for clinical TRAb type detection, and the method can be used for developing specific therapeutic drugs for stimulating TRAb antibodies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-thyroid stimulating hormone receptor monoclonal antibody 1A4 and an application thereof. Background Art

[0002] Graves' disease (GD) is an autoimmune thyroid disease caused by the interaction of natural, environmental and genetic factors. Thyroid stimulating hormone receptor (TSHR) antibodies (TRAb) are the core mechanism of GD pathogenesis. TRAb is mainly an autoantibody produced against the thyroid stimulating hormone receptor (TSHR).

[0003] TRAb not only plays a core role in the pathogenesis of GD, but is also closely related to the prognosis and recurrence of the disease. Accurate detection of TRAb levels is a key indicator for predicting the course of GD at the beginning of diagnosis and at different stages of treatment, guiding treatment and preventing recurrence. With the vigorous development of biological therapy, hyperthyroidism caused by autoimmune thyroid disease has attracted much attention, and research on TRAb-related antisense peptides and monoclonal antibodies is a hot topic in this professional academic field.

[0004] TRAb is an oligoclonal antibody IgG produced in GD patients. It was first discovered in GD hyperthyroidism patients in 1956 and was initially called a long-acting thyroid stimulator. TRAb is a polyclonal antibody, which is mainly divided into stimulating antibodies (TSAb), blocking antibodies (TBAb) and neutral antibodies. Among them, TSAb mainly binds to TSHR to initiate the second signal, which plays a major and 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 hypothyroidism in GD patients in the later stage. Neutral antibodies binding to TSHR may induce thyroid apoptosis and oxidative stress. At present, although bioanalysis has been developed and bridging technology has been used to analyze and detect TRAb, the detection of TRAb in clinical practice still uses traditional competitive assays. However, this competitiveness can only determine the presence of TRAb in the serum of GD patients, and cannot completely distinguish TSAb, TBAb and neutral antibodies. The other two detection methods have not been widely used in clinical detection due to their long cycle and high detection components. This results in clinicians being unable to accurately judge whether GD patients are in the active phase or remission phase when diagnosing GD patients, and being unable to provide individualized and precise treatment for GD patients, which may lead to delays and aggravation of the patient's condition.

[0005] At present, the clinical treatment of GD hyperthyroidism still relies on three methods that have been used for decades: antithyroid drugs (ATDs), 131 I treatment and thyroidectomy. These treatments can only improve the thyroid function of some patients in the short term, and most patients' autoimmunity will soon reactivate, and serum TRAb will reappear or even be higher than before.131 After I treatment, the serum TRAb level of patients will surge in the short term, and may even cause GD eye disease and accelerate its progression. The fundamental reason is that these treatments do not fundamentally solve the problem of abnormal production of TRAb in GD patients, but only provide short-term relief by inhibiting the production of thyroid hormones, or the patient progresses to the remission period of the GD disease itself. 131 I treatment may damage the body's immunity, leading to excessive inhibition of T cell activation, reduced immune active cells, and uncontrolled antibody release. At present, emerging targeted drugs such as lymphocyte-targeted biological agents (rituximab, RTX), small molecule TSHR antagonists (ANTAG-3, VA-K-14 and S37a), TBAb active targeted drugs (K1-70) and TSHR mixed peptides (ATX-GD-59) have also been confirmed to have no significant effect on GD patients.

[0006] Based on the crucial role of TRAb in the development of GD and GD-related diseases, treatment methods targeting TRAb have received increasing attention. Pathological stimulatory TRAb (TSAb) competes with the natural ligand TSH to bind to TSHR, initiates the signaling pathway to produce cyclic adenosine monophosphate (cAMP), and stimulates thyroid hormone synthesis and secretion, which is the main pathogenesis of GD hyperthyroidism. TSAbs competitive antagonists - TSHR monoclonal antibodies (MAbs) have always attracted much attention as potential therapeutic drugs for GD, and the focus of all the above studies is TSHR and TRAb.

[0007] TSHR is a member of the G protein-coupled receptor superfamily. It exists in the plasma membrane of thyroid follicular epithelial cells. It has a total length of 764 amino acid residues, including a large extracellular region (ECD), a seven-helix transmembrane domain (TMD) and a C-terminal tail. The ECD contains the TSHR leucine-rich domain (LRD) and hinge region, binds to TSAb, and activates the G protein through the conformational change of the TMD. After removing the first 21 signal peptides, the ECD contains 22-410 amino acid residues and has 6 glycosylation sites. After TSHR transcription and translation, it will be cleaved into A subunit and B subunit linked by 2 disulfide bonds. Among them, the A subunit is a secreted, soluble, heavily glycosylated protein containing TSHR amino acid residues 22-289, located in the ECD and covering all glycosylation sites. The TSHR A subunit mainly mediates the maturation of TRAb with high affinity to TSHR in GD patients. By immunizing mice with adenovirus encoding the human TSHR gene, it was found that the shed A subunit can induce hyperthyroidism in most mice, and an increase in TRAb can be detected in the serum of mice. The shed A subunit may be released into thyroid lymphocytes and captured by antigen-presenting cells. The mannose receptors expressed on these cells bind to the highly glycosylated A subunits, are processed into polypeptides and presented to T cells, which may in turn lead to TSAb reactions. Therefore, the TSHR A subunit is currently considered to be the main immunogen of GD.

[0008] The preparation of monoclonal antibodies using TSHR A subunit as the main immunogen may help deepen the exploration of TSAb structure and function. Although different laboratories have used different methods and different TSHR antigen fragments to immunize mice and hamsters to prepare animal-derived monoclonal antibodies and directly isolated and cloned human-derived monoclonal antibodies from human peripheral blood. However, these have not changed the embarrassing situation of difficult diagnosis and treatment of GD in clinical practice. GD is still a refractory disease so far. The present invention attempts to screen and use antigen fragments of the main pathogenic sites of TRAb to prepare targeted and active TSHR MAbs, and establish specific detection methods and treatment methods for corresponding autoantibodies using these TSHR MAbs as targets, which will play an active role in predicting the course of GD, guiding treatment and preventing recurrence, and also has far-reaching significance for exploring the pathological mechanism of GD. Summary of the invention

[0009] In view of the above technical problems, the purpose of the present invention is to provide an anti-thyroid stimulating hormone receptor monoclonal antibody 1A4 and its application. Specifically, it includes the following contents:

[0010] In a first aspect, the present invention 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;

[0011] The variable region CDR of the antibody heavy chain includes 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;

[0012] The variable region CDR of the antibody light chain includes 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.

[0013] Preferably, the variable region FR of the antibody heavy chain includes 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;

[0014] The variable region FR of the antibody light chain includes FR1 as shown in the amino acid sequence of SEQ ID NO.13, FR2 as shown in the amino acid sequence of SEQ ID NO.14, FR3 as shown in the amino acid sequence of SEQ ID NO.15 and FR4 as shown in the amino acid sequence of SEQ ID NO.16.

[0015] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown as SEQ ID NO.8, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NO.17.

[0016] In a second aspect, the present invention provides a nucleic acid encoding the antibody heavy chain and antibody light chain of the monoclonal antibody 1A4 described in the first aspect.

[0017] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown as SEQ ID NO.9, and the nucleotide sequence encoding the variable region of the antibody light chain is shown as SEQ ID NO.18.

[0018] In a third aspect, the present invention provides use of the monoclonal antibody 1A4 described in the first aspect in the preparation of a specific therapeutic drug for anti-irritant TRAb.

[0019] Preferably, the specific therapeutic drug is an immunosuppressant.

[0020] In a fourth aspect, the present invention provides use of the monoclonal antibody 1A4 described in the first aspect in preparing a reagent or a kit for detecting TRAb.

[0021] In a fifth aspect, the present invention provides use of the monoclonal antibody 1A4 described in the first aspect in preparing a diagnostic or predictive kit for a disease associated with TSHR.

[0022] Preferably, the TSHR-related disease is autoimmune thyroid disease.

[0023] Preferably, the autoimmune thyroid disease comprises Graves' disease.

[0024] The beneficial effects of the present invention are as follows: ① The present invention first prepares a recombinant TSHR289 protein, immunizes mice with the recombinant TSHR289 protein, and obtains an anti-thyroid stimulating hormone receptor monoclonal antibody 1A4, wherein 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; ② The monoclonal antibody 1A4 has a high affinity with the recombinant TSHR289 protein and can bind to the natural wild-type TSHR, indicating that the monoclonal antibody 1A4 is an antibody with a conformational epitope. Most of the current mouse-derived anti-TSHR289 monoclonal antibodies are linear and have no biological properties, while the monoclonal antibody 1A4 described in the present application has a high affinity with the natural wild TSHR, and the antibody has a conformational structure and biological properties; ③ The monoclonal antibody 1A4 can competitively inhibit the binding of TRAb and TSHR in the serum of GD patients, has a strong biological activity, and is clinically useful. The detection of TRAb provides a theoretical basis and a powerful tool; ④ the monoclonal antibody 1A4 has high in vitro and in vivo thyroid stimulating activity. Compared with the mouse anti-TSHR monoclonal antibody reported in the existing literature, the monoclonal antibody 1A4 described in the present application has stronger thyroid stimulating activity; ⑤ the monoclonal antibody 1A4 can competitively inhibit the binding of TRAb and TSHR in GD hyperthyroidism patients. Even when the patient's serum is diluted to 16 times, it can still effectively inhibit the binding. The inhibitory activity is higher than that of the relevant mouse antibodies reported in the existing literature. It is possible to be used as an anti-TSHR monoclonal antibody passive immunization preparation product or to develop an in vitro diagnostic kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Purity analysis results of monoclonal antibody 1A4; wherein, M is a protein marker, lane 1 is the purified anti-TSHR monoclonal antibody 1A4-IgG of reduced type, and lane 2 is the purified anti-TSHR monoclonal antibody 1A4-IgG of non-reduced type;

[0026] Figure 2 Affinity test results of monoclonal antibody 1A4;

[0027] Figure 3 Results of the binding analysis of monoclonal antibody 1A4 and wild-type TSHR;

[0028] Figure 4 Activity analysis results of monoclonal antibody 1A4;

[0029] Figure 5 Competitive ELISA was used to detect the ability of HRP-labeled monoclonal antibody 1A4 to competitively inhibit the binding of TRAb in GD patient serum to TSHR; A: 1A4 was diluted in multiple ratios (2000-fold, 4000-fold, 6000-fold, and 8000-fold dilutions), and ELISA was used to detect the ability to competitively inhibit the binding of TRAb in GD patient serum to TSHR, and the highest dilution of 1A4 was determined; B: 1A4 was diluted at a ratio of 1:8000, and ELISA was used to detect the ability to competitively inhibit the binding of 32 GD patient serum and 14 normal healthy patient serum to TSHR; C: 1A4 was diluted at a ratio of 1:8000, and ELISA was used to detect the ability to competitively inhibit the binding of TRAb in GD patient serum to TSHR after multiple dilutions;

[0030] Figure 6 Results of in vivo activity detection of monoclonal antibody 1A4; A is the TT4 detection of mouse serum in the control group and the monoclonal antibody 1A4 immunization group; B is the HE section of the thyroid gland of mice in the control group, I is under a 10× microscope, and II is under a 40× microscope; C is the HE section of the thyroid gland of mice in the monoclonal antibody 1A4 immunization group, both under a 40× microscope, the red arrow indicates follicular epithelial cell hyperplasia, and the black arrow indicates follicular expansion. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0032] The 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 Laboratory of Lanzhou Institute of Biological Products Co., Ltd.; the TSHR289 construct was amplified using the first 289 amino acid residues of human TSHR as a template and cloned into pFastBac1 constructed by Nanjing GenScript Biotechnology Co., Ltd.; adenovirus expressing human TSHR289 was constructed by Shanghai Jikai Co., Ltd.; sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd.; plasmid extraction kit was purchased from Qiagen; lip3000 was purchased from Thermo Fisher Scientific; PEG1450 was purchased from Beijing Bioson Biotechnology Co., Ltd.; Sf-900TMII SFM medium, 1640 medium, F12K medium, fetal bovine serum, HT and HA T were all 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 Sino-Bio; affinity purification medium was purchased from GE; fluorescence microscope was purchased from ZEISS; ELISA multiwell plate reader was purchased from Molecular Devices; ELISA plate washer was purchased from BIO-RAD; flow cytometry instrument was purchased from BD; cAMP detection kit was purchased from R&D.

[0033] Example 1 Preparation of monoclonal antibody 1A4

[0034] 1. Expression of recombinant TSHR289 protein

[0035] The recombinant pFastBacTM1 plasmid TOP10 E.coli containing the TSHR289 gene (expressing the first 289 amino acid residues of human TSHR) was inoculated into LB medium for overnight culture. The target plasmid was extracted and transformed into DH10 Bac E.coli. After inoculation into SOC medium for culture, it was inoculated into a screening plate for overnight culture. The white monoclonal colonies with good growth were selected, inoculated into SOC medium for overnight culture, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The ones with correct sequencing were expanded and the plasmid was extracted. The recombinant Bacmid DNA was transfected into sf9 cells with good growth using lip3000, and the primary virus was obtained in one week. The cell supernatant was collected, centrifuged at 1000g for 5min, and filtered through a 0.22um filter membrane to obtain the virus. The primary virus continued to infect sf9 cells to expand the virus amount. The virus was inoculated into High FiveTM cells at an MOI of 5pfu / cell, and the optimal protein harvest time was obtained 72h after infection. The expression of target protein TSHR289 was detected by Western blot, and the recombinant protein TSHR289 was purified by NI column affinity chromatography. The purity of the recombinant target protein TSHR289 was detected by SDS-Page.

[0036] 2. Mouse Immunization

[0037] Human TSHR 289 (recombinant TSHR289 protein) expressed by the adenovirus was injected into the left and right femoral muscles of 6-8 week old female BALB / c mice, immunized once every 3 weeks, for 3 consecutive times. Blood was collected from the tail vein one week after the second and third immunizations to detect serum titers, and mice with higher titers received the third and booster immunizations, respectively.

[0038] 3. Serum titer determination

[0039] Blood was collected from the tail vein at different time periods, incubated at 37°C for 1 hour, incubated at 4°C for 2 hours, and centrifuged at 4000rpm for 30 minutes to obtain serum. The serum was diluted in multiples from 1:500 to 1:160000. The purified TSHR289 recombinant protein was diluted to 5ug / mL with PBS, 100μL / well was coated on the ELISA plate, and blocked overnight. 100μL / well was added with the mouse serum diluted in multiples (incubated at 37°C for 1h). The diluent was discarded, the plate was washed 5 times with PBST solution, and HRP-labeled anti-mouse IgG enzyme-labeled secondary antibody was added (incubated at 37°C for 1h). The diluent was discarded, the plate was washed 5 times with PBST solution, and the A and B mixtures were added. After incubation at 37°C for 15 minutes, 50μL of stop solution was added to each well, and the OD was read by the ELISA plate reader. 450 Numeric value.

[0040] 4. Preparation of Hybridomas

[0041] Before fusion, 6-8 week old BALB / c female mouse peritoneal macrophages were used to prepare feeder cells. The mouse spleen cells with the highest antibody titer were selected to fuse with the SP2 / 0 osteomyeloma cells in good growth state, and PEG1450 was added at a ratio of 10:1 for fusion. After the fusion was completed, HAT screening medium containing 20% ​​FBS was added to gently resuspend the fused cells, and 150 μL was added to each well of the 96-well cell culture plate with feeder cells. Place in a 37°C, 5% CO2 cell culture incubator for culture, and observe the growth of the cells the next day. Two days after fusion, half of the medium was replaced with 20% FBS HAT selection medium. On the fourth day after fusion, the cell fusion was observed under a microscope, and the culture wells with hybridoma cell clones were marked.

[0042] 5. Screening of positive hybridoma cells and their cloning culture

[0043] The hybridoma cell culture supernatant was tested by full-plate ELISA. The first round of screening used indirect ELISA to screen positive hybridoma cell clones. The primary antibody was the hybridoma cell culture supernatant, and the secondary antibody was HRP-labeled goat anti-mouse enzyme-labeled secondary antibody. Try to choose the absorbance OD 450 Positive hybridoma cells with high read values, small number of cell clones, and good cell status are cloned and cultured. The limiting dilution method is used for 3-5 rounds of screening. In each round of cloning, the positive monoclonal hybridoma cells are expanded and subcultured. During the subculture, indirect ELISA tests are also performed to detect the cell subculture stability, and the cells are kept frozen.

[0044] 6. Large-scale preparation and purification of monoclonal antibodies

[0045] Positive monoclonal hybridoma cells were transferred to 24-well cell culture plates for culture, and then transferred to 6-well cell culture plates, T25 and T75 cell culture flasks for expansion. When the cells in the cell culture flasks rapidly proliferated, the cultured cells were collected, centrifuged at 1000 rpm for 5 min, and 1×10 6 The cells were intraperitoneally injected into pre-sensitized BALB / c female mice. One week later, the mouse ascites was collected, centrifuged at 12000rpm for 30min, resuspended in PBS and filtered with a 0.45um filter membrane. The mouse hybridoma antibody was purified using a Protein G pre-packed column. The protein concentration of the monoclonal antibody was determined using the GE NanoVue ultra-micro spectrophotometer Protein A280 method. Sterile filtration was performed and stored at 2-8°C. The anti-TSHR monoclonal antibody obtained by screening was numbered 1A4; the sequencing results showed that the amino acid sequences of CDR1, CDR2, and CDR3 in its heavy chain variable region were:

[0046] CDR1: GFSLTGYG (shown in SEQ ID NO. 1);

[0047] CDR2: IWGDGST (shown in SEQ ID NO. 2);

[0048] CDR3: AREREDGYYDAMDY (shown in SEQ ID NO. 3);

[0049] The amino acid sequences of the four FR regions of the heavy chain variable region are:

[0050] FR1: EVQLEESGPGLVAPSQSLSITCTVS (shown in SEQ ID NO. 4);

[0051] FR2: VNWVRQPPGKGLEWLGM (shown in SEQ ID NO.5);

[0052] FR3: DYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYC (shown in SEQ ID NO. 6); FR4: WGQGTSVTVSS (shown in SEQ ID NO. 7);

[0053] The amino acid sequence of the heavy chain variable region: EVQLEESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPP GKGLEWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARERED GYYDAMDYWGQGTSVTVSS (shown in SEQ ID NO.8);

[0054] Nucleotide sequence of the heavy chain variable region: GAGGTGCAGCTGGAGGAGGTCAGGACCTGGCCTGGTGGC GCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGGGTGATGGAAGCACAGACTATAATTCAGCTCTCAAATCCAGACTG AGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAGAGAGAGGATGGTTACTACGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.9 shown).

[0055] The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are:

[0056] CDR1: QSLLYSSNQKNY (shown in SEQ ID NO. 10);

[0057] CDR2: WAS (shown in SEQ ID NO. 11);

[0058] CDR3: QQYYSYPR (shown in SEQ ID NO. 12);

[0059] The amino acid sequences of the four FR regions of the light chain variable region are:

[0060] FR1: DIVMTQSPSSLAVSVGEKVTMSCKSS (shown in SEQ ID NO. 13);

[0061] FR2: LAWFQQNPGQSPKLLIY (shown in SEQ ID NO.14);

[0062] FR3: TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC (shown in SEQ ID NO. 15);

[0063] FR4: GRSVEAPSWKSN (shown in SEQ ID NO. 16);

[0064] The amino acid sequence of the light chain variable region: DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLA WFQQNPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYP RGRSVEAPSWKSN (shown in SEQ ID NO.17);

[0065] Nucleotide sequence of the light chain variable region: GACATTGTGATGACCCAGTCTCCATCCTCCCTAGCTGTGT CAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTTCCAGCAGAATCCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCC CTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATAGCTATCCACGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC(SEQ IDNO.18).

[0066] Example 2: Detection of properties of monoclonal antibody 1A4

[0067] 1. Antibody Purity Analysis

[0068] In this example, the anti-TSHR monoclonal antibody 1A4 was prepared in large quantities by cell culture method and mouse ascites method. The cell culture supernatant and the ascites after crude extraction were treated with Protein G. After the sample was eluted, the protein concentration was measured by NanoVue ultra-micro spectrophotometer to be 430 μg / ml. Since the molecular weight of the monoclonal antibody is relatively large (mostly 150-180 kDa), it was reduced with β-mercaptoethanol, and the purity of the purified monoclonal antibody was analyzed by reducing and non-reducing SDS-PAGE electrophoresis.

[0069] The results are as follows Figure 1 As shown in the figure, the monoclonal antibody is reduced to 55kDa (heavy chain) and 25kDa (light chain) by β-mercaptoethanol reducing agent, and the molecular weight of the non-reduced monoclonal antibody is about 150kDa, which is consistent with the molecular weight of IgG. The electrophoresis results show that the protein G medium affinity purification method has obtained a high-purity monoclonal antibody, and the antibody purity has reached more than 90%.

[0070] 2. Antibody affinity testing

[0071] This example uses surface plasmon resonance analysis to detect the affinity kinetic properties of anti-TSHR monoclonal antibody 1A4 and TSHR289 fusion protein. A 10mM sodium acetate solution with a pH of 4.5 was used as the coating solution, the CM5 chip was activated under the action of EDC / NHS, ethanolamine was used for blocking, the coating flow rate was 10μL / min, the running buffer was HBS-EP buffer, and the purified TSHR289 fusion protein was coated on the CM5 chip. The monoclonal antibody 1A4 was diluted to 3.0μg / mL with HBS-EP Buffer, and it was flowed through the CM5 chip coated with purified TSHR289 in turn, with an injection flow rate of 30μL / min. After the binding was completed, it was regenerated in a Gly-HCL buffer with a pH of 1.5. After the reaction was completed, Biacore T200 Evaluation Software 1.0 was used for analysis.

[0072] The results are as follows Figure 2 As shown, affinity assay showed that the affinity of purified TSHR289 protein to monoclonal antibody 1A4 was 6.333×10 10 .

[0073] 3. Analysis of antibody binding to wild-type TSHR

[0074] In this example, flow cytometry was used to detect the binding ability of anti-TSHR monoclonal antibody 1A4 to wild-type human TSHR stably transfected in CHO cells. CHO cells stably transfected with wild-type human TSHR in good growth state were digested with trypsin, counted, centrifuged at 1000 rpm for 5 minutes, and resuspended in 2% BSA. The Fab segment binding test of monoclonal antibody 1A4 was performed under the same conditions after adding Fcblock. 1×10 6 10 μg of purified monoclonal antibody 1A4 was added to each tube and incubated at room temperature in the dark for 60 min. At the same time, an equal volume of PBS was added to the control group. The cells were washed 3 times with 2% BSA, and the secondary antibody labeled with Per-CP was added and incubated at room temperature in the dark for 45 min. The cells were washed 3 times with 2% BSA and resuspended. The cells were detected and analyzed using a BD AccuriC6 flow cytometer, and the results were analyzed using flowjo software.

[0075] The results are as follows Figure 3 As shown, the binding rates of the IgG and Fab fragments of the anti-TSHR monoclonal antibody 1A4 to the wild-type TSHR were 76.3% and 82.3%, respectively.

[0076] 4. Antibody Activity Analysis

[0077] In this example, competitive ELISA was used to detect the activity of anti-TSHR monoclonal antibody 1A4 in stimulating CHO cells stably transfected with wild-type human TSHR to produce cAMP. CHO cells stably transfected with wild-type human TSHR in good growth state were digested with trypsin and the cells were counted at 5×10 4 Cells / well were evenly plated in a 96-well cell culture plate, and the cell adhesion was observed after culturing in F12K complete medium for 24 hours. After the cells adhered well, the original medium was discarded, and after culturing in serum-free medium for 2 hours, purified monoclonal antibody 1A4 diluted in different concentrations containing 1% BSA and 0.5mM 3-isobutyl-1-methylxanthine was added to each well and incubated at 37°C for 4 hours. Washed 3 times with PBS, 150μL / well of cell lysate was added to the 96-well plate, repeatedly frozen and thawed 3 times at -80°C, and finally overnight at -80°C. The next day, the test was performed according to the method of the ELISA kit manufacturer, and the results were expressed in pmol / ml.

[0078] The results are as follows Figure 4 As shown, the EC values ​​of IgG and Fab fragments of anti-TSHR monoclonal antibody 1A4 are 50 They are 0.75μg / ml and 0.47μg / ml respectively.

[0079] 5. Analysis of competition between antibodies and TRAb in serum of GD patients for receptor binding

[0080] This example uses competitive ELISA technology to detect the ability of anti-TSHR monoclonal antibody 1A4 to inhibit the binding of TRAb to TSHR in human serum. 46 human serum samples were collected, including sera from 32 TRAb-positive patients with clinically diagnosed GD hyperthyroidism and sera from 14 TRAb-negative normal healthy people (without any other diseases including any autoimmune diseases). Take 100 μL of serum sample and add it to the ELISA plate coated with TSHR antigen included in the commercial kit. After incubation at 37°C for 60 minutes, wash the plate 5 times with PBST, add 100 μL of HRP-labeled 1A4 diluted at different concentrations, incubate at 37°C for 60 minutes, wash the plate 5 times with PBST, add the mixed A+B solution, incubate at 37°C for 15 minutes, add the color developing solution C, and shake the microplate at OD 450 nm reading. In addition, the serum samples were diluted and tested in the same way. The inhibition rate was calculated as follows: Inhibition rate (%) = (B0-Bi) / B0 × 100%. Bi is the OD value of the experimental sample, and B0 is the OD value of the control sample.

[0081] The results are as follows Figure 5The results show that A: 1A4 was diluted in multiples (2000 times, 4000 times, 6000 times, and 8000 times), and the ability of competitive inhibition of TRAb binding to TSHR in serum of GD patients was detected by ELISA to determine the highest dilution of 1A4; B: 1A4 was diluted at a ratio of 1:8000, and the ability of competitive inhibition of 32 GD patients and 14 normal healthy patients' sera binding to TSHR was detected by ELISA; C: 1A4 was diluted at a ratio of 1:8000, and the ability of competitive inhibition of TRAb binding to TSHR in serum of GD patients after multiple dilution was detected by ELISA. The results show that the monoclonal antibody 1A4 described in the present application can still effectively inhibit the binding of human serum TRAb to TSHR on the coated plate at a concentration of 8000 times dilution, and can inhibit the binding of most GD patient serum TRAb (with an inhibition rate of 50% as the inhibition standard). And it can inhibit the binding of GD patient serum to TSHR when it is diluted 16 times.

[0082] 6. Antibody activity detection in vivo

[0083] This example uses ELISA technology and HE sections to detect the biological activity of anti-TSHR monoclonal antibody 1A4 in animals. The purified monoclonal antibody 1A4 was intraperitoneally injected into 6-week-old BALB / c female mice at a dose of 250 μg, twice, once every 3 days, and the control group was immunized in the same way with PBS. After 2 days, blood was collected and the mice were killed. The thyroid gland of the mice was taken, fixed, dehydrated, embedded and sectioned for HE staining.

[0084] The results are as follows Figure 6 As shown, the serum TT4 level was (2.5±0.5) ng / ml (MEAN±SEM; n=12), which was not statistically significant compared with the control mice (2.5±0.5 ng / ml) ( Figure 6 However, HE sections showed mild hyperplasia of mouse thyroid follicular epithelial cells, with different follicle shapes and sizes, and follicular expansion ( Figure 6 B and C).

[0085] The above activity analysis results show that:

[0086] The present invention successfully prepared the monoclonal antibody 1A4 against TSHR289 protein, which not only has high affinity with the purified TSHR289 protein, but also can bind to the natural wild-type TSHR, which suggests that it is an antibody with a conformational epitope. Secondly, the monoclonal antibody 1A4 also has thyroid stimulating activity, and can stimulate CHO cells transfected with the human TSHR full-length receptor to produce cAMP even at a lower concentration (μg / ml level). The thyroid stimulating activity of the monoclonal antibody 1A4 described in the present invention is similar to the activity of TRAb in human serum currently reported. At the same time, the present invention also detected that the Fab segment of the monoclonal antibody 1A4 also exhibited similar activity.

[0087] The present invention uses HRP to label monoclonal antibody 1A4, and competitive ELISA detection finds that monoclonal antibody 1A4 after 8000 times dilution can also competitively inhibit the binding of TRAb and TSHR in the serum of GD patients, and this conclusion is also verified in the serum of 32 TRAb-positive GD patients. After diluting the serum of GD patients, it is found that monoclonal antibody 1A4 can inhibit the binding of TRAb and TSHR in the serum diluted 16 times, indicating that monoclonal antibody 1A4 has strong biological activity, which provides a theoretical basis and powerful tool for the current clinical detection of TRAb.

[0088] After immunizing female BALB / c female mice with the purified monoclonal antibody 1A4 of the present invention, there was no significant change in serum TT4 level compared with the control group PBS immunization group. However, mild hyperplasia of follicular epithelial cells was found on the thyroid gland of the immunized mice, the follicles were of different sizes, and the follicles showed typical pathological changes of expansion. This shows that although the typical hyperthyroidism manifestations of mice could not be induced, the monoclonal antibody 1A4 still showed a certain thyroid stimulating activity in vivo.

[0089] In summary, the monoclonal antibody 1A4 described in the present invention has high thyroid stimulating activity in vitro and in vivo, and can competitively inhibit the binding of TRAb and TSHR in patients with GD hyperthyroidism. Compared with the currently reported mouse-derived anti-TSHR289 monoclonal antibody, ① the monoclonal antibody 1A4 described in the present application has a high affinity with the natural wild TSHR, and the antibody has a conformational structure and biological characteristics; ② it has stronger thyroid stimulating activity; ③ it has a stronger ability to competitively inhibit the binding of TRAb and TSHR in patients with GD hyperthyroidism. It is possible to be used as an anti-TSHR monoclonal antibody passive immunization preparation product or to develop an in vitro diagnostic kit.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-thyroid stimulating hormone 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 includes 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 includes 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 according to claim 1, characterized in that The variable region FR of the antibody heavy chain includes 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 includes FR1 as shown in the amino acid sequence of SEQ ID NO.13, FR2 as shown in the amino acid sequence of SEQ ID NO.14, FR3 as shown in the amino acid sequence of SEQ ID NO.15 and FR4 as shown in the amino acid sequence of SEQ ID NO.

16.

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

17.

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

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

18.

6. Use of the monoclonal antibody 1A4 according to any one of claims 1 to 3 in the preparation of a specific therapeutic drug for anti-irritant TRAb.

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

8. Use of the monoclonal antibody 1A4 according to any one of claims 1 to 3 in the preparation of a diagnostic or predictive kit for a disease associated with TSHR.

9. The use according to claim 8, characterized in that The TSHR-related disease is an autoimmune thyroid disease.

10. The use according to claim 9, characterized in that The autoimmune thyroid diseases include Graves' disease.

Images