A monoclonal antibody against thyroid-stimulating hormone receptor 22B1 and its application

Abstract

This invention relates to the field of biotechnology, specifically to an anti-thyroid-stimulating hormone receptor monoclonal antibody 22B1 and its applications. The invention discloses an anti-thyroid-stimulating hormone receptor monoclonal antibody 22B1, the sequence of which includes the heavy chain variable region shown in SEQ ID NO. 8 and the light chain variable region shown in SEQ ID NO. 17. The monoclonal antibody 22B1 exhibits high affinity for TSHR289 protein, can bind to natural wild-type TSHR, possesses thyroid-stimulating activity, and can be competitively inhibited by TRAb in the serum of GD patients, thus exhibiting strong biological activity. It can be used in the preparation of specific therapeutic drugs against stimulating TRAb or in the development of in vitro diagnostic kits, providing a theoretical basis and powerful tool for the clinical detection of TRAb types, and can also be used to develop specific therapeutic drugs against stimulating TRAb antibodies.

Description

[0001] This application claims priority to the earlier application filed on September 27, 2024, with application number CN2024113608004 and entitled "An anti-thyroid-stimulating hormone receptor monoclonal antibody 22B1 and its application", the entire contents of which are set forth in this application. Technical Field

[0002] This invention relates to the field of biotechnology, specifically to an anti-thyroid-stimulating hormone receptor monoclonal antibody 22B1 and its applications. Background Technology

[0003] Graves' disease (GD) is an autoimmune thyroid disease caused by the interaction of natural, environmental and genetic factors. Thyroid-stimulating hormone receptor (TSHR) antibody (TRAb) is the core mechanism of GD pathogenesis. It is an autoantibody produced mainly against the thyroid-stimulating hormone receptor (TSHR).

[0004] TRAb plays a central role in the pathogenesis of Graves' disease (GD) and is closely related to the disease's outcome and recurrence. Accurate detection of TRAb levels is a key indicator for predicting disease progression, guiding treatment, and preventing recurrence at the initial diagnosis and different stages of treatment for GD. With the rapid development of biotherapy, hyperthyroidism caused by autoimmune thyroid diseases has received considerable attention, and research on TRAb-related antisense peptides and monoclonal antibodies is a hot topic in this field.

[0005] TRAb is an oligoclonal IgG antibody produced in patients with Graves' disease (GD). It was first discovered in GD hyperthyroidism patients in 1956 and initially referred to as a long-acting thyroid stimulator. TRAb is a polyclonal antibody, mainly divided into stimulating antibodies (TSAb), blocking antibodies (TBAb), and neutral antibodies. TSAb primarily binds to TSHR to initiate the second signal, playing a major decisive role in the pathogenesis of GD hyperthyroidism. TBAb is thought to play a major role during disease remission and may be a cause of hypothyroidism in later stages of GD. Neutral antibodies binding to TSHR may induce thyroid apoptosis and oxidative stress. Currently, although bioanalytical methods and bridging techniques have been developed to analyze and detect TRAb, traditional competitive assays are still used clinically for TRAb detection. However, this competitive assay can only confirm the presence of TRAb in the serum of GD patients and cannot completely distinguish between TSAb, TBAb, and neutral antibodies. The other two detection methods, due to their long processing times and high detection requirements, have not been widely adopted in clinical testing. This makes it difficult for clinicians to accurately determine whether a patient with Gram-D disease is in an active or remission phase, hindering the implementation of individualized and precise treatment and potentially leading to delays and worsening 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 is a member of the G protein-coupled receptor superfamily and is located in the plasma membrane of thyroid follicular epithelial cells. It consists 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 the TSHR leucine-rich domain (LRD) and hinge region, which bind to TSAb and activate G proteins through conformational changes in the TMD. After removing the first 21 signal peptides, the ECD contains 22-410 amino acid residues and has 6 glycosylation sites. Following transcription and translation, TSHR cleaves into an A subunit and a B subunit linked by two disulfide bonds. The A subunit is a secreted, soluble, heavily glycosylated protein containing TSHR amino acid residues 22-289, located within the ECD, and encompassing all glycosylation sites. The TSHR A subunit primarily mediates the maturation of TRAb with high affinity for TSHR in Gram-Disease (GD) patients. Immunization of mice with an adenovirus-encoded human TSHR gene revealed that the shed A subunit induced hyperthyroidism in most mice, and elevated TRAb levels were detected in the mouse serum. The shed A subunit may be released into thyroid lymphocytes and captured by antigen-presenting cells. The mannose receptor expressed on these cells binds to the highly glycosylated A subunit, which is then processed into a peptide and presented to T cells, potentially leading to a TSAb response. Therefore, the TSHR A subunit is currently considered a major immunogen in GD.

[0009] Preparing monoclonal antibodies using TSHR A subunits as the primary immunogen may help deepen the understanding of TSAb structure and function. Although different laboratories have prepared animal-derived monoclonal antibodies by immunizing mice and hamsters with different methods and TSHR antigen fragments, and human-derived monoclonal antibodies directly isolated and cloned from human peripheral blood, these have not changed the difficult situation of diagnosing and treating Graves' disease (GD) in clinical practice. GD remains a refractory disease. This invention attempts to screen and use antigen fragments of the main pathogenic sites of TRAb to prepare targeted and relatively active TSHR MAbs. Using these TSHR MAbs as targets, specific detection and treatment methods for corresponding autoantibodies can be established, which will play a positive role in predicting the course of GD, guiding treatment, and preventing relapse. It also has profound significance for exploring the pathological mechanisms of GD. Summary of the Invention

[0010] To address the aforementioned technical problems, the present invention aims to provide an anti-thyroid-stimulating hormone receptor monoclonal antibody 22B1 and its applications. Specifically, it includes the following:

[0011] In a first aspect, the present invention provides an anti-thyroid-stimulating hormone receptor monoclonal antibody 22B1, wherein the monoclonal antibody 22B1 comprises an antibody heavy chain and an antibody light chain;

[0012] 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;

[0013] 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.

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

[0015] The variable region FR of the antibody light chain includes 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 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.

[0017] In a second aspect, the present invention provides a nucleic acid that encodes the antibody heavy chain and antibody light chain of the monoclonal antibody 22B1 described in the first aspect above.

[0018] Preferably, 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.

[0019] Thirdly, the present invention provides the use of the monoclonal antibody 22B1 described in the first aspect above in the preparation of a specific therapeutic agent for the anti-irritant TRAb.

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

[0021] Fourthly, the present invention provides the use of the monoclonal antibody 22B1 described in the first aspect above in the preparation of reagents or kits for detecting TRAb.

[0022] Fifthly, the present invention provides the use of the monoclonal antibody 22B1 described in the first aspect above in the preparation of diagnostic or predictive kits for diseases related to TSHR.

[0023] Preferably, the disease associated with TSHR is an autoimmune thyroid disease.

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

[0025] The beneficial effects of this invention are as follows: ① This invention first prepared recombinant TSHR289 protein, immunized mice with the TSHR A subunit encoded by adenovirus, and used the purified recombinant TSHR289 protein as a screening antigen to screen and obtain an anti-thyroid-stimulating hormone receptor monoclonal antibody 22B1. The monoclonal antibody 22B1 contains the heavy chain variable region shown in SEQ ID NO.8 and the light chain variable region shown in SEQ ID NO.17; ② The monoclonal antibody 22B1 has a high affinity for recombinant TSHR289 protein and can also bind to natural wild-type TSHR; ③ The monoclonal antibody 22B1 can be competitively inhibited by TR Ab in the serum of a small number of GD patients from binding to TSHR, exhibiting human-like TRAb biological characteristics, providing a theoretical basis and powerful tool for clinical TRAb detection; ④ The monoclonal antibody 22B1 has high in vitro and in vivo thyroid-stimulating activity and may be used as a passive immunization agent product against TSHR monoclonal antibody or for developing in vitro diagnostic kits. Attached Figure Description

[0026] Figure 1 The purity analysis results of monoclonal antibody 22B1; where M is the protein marker, lane 1 is the purified anti-TSHR monoclonal antibody 22B1-IgG in reduced form, and lane 2 is the purified anti-TSHR monoclonal antibody 22B1-IgG in non-reduced form.

[0027] Figure 2 Affinity test results for monoclonal antibody 22B1;

[0028] Figure 3 Results of binding affinity analysis between monoclonal antibody 22B1 and wild-type TSHR;

[0029] Figure 4 Results of activity analysis of monoclonal antibody 22B1;

[0030] Figure 5 Analysis results of serum TRAb competitive inhibition monoclonal antibody 22B1 binding to receptor in GD patients;

[0031] In this study, A: 22B1 was serially diluted (2000-fold, 4000-fold, and 8000-fold), and the ability of 22B1 to bind to TSHR was detected by ELISA to determine the optimal dilution of 22B1; B: 22B1 was diluted at the optimal dilution ratio, and ELISA was used to detect the competitive inhibition of TRAb binding of 22B1 to the TSHR receptor in the serum of 32 GD patients, with serum from 14 healthy patients serving as a control.

[0032] Figure 6 Results of in vivo activity assay of monoclonal antibody 22B1; A shows the detection of serum TT4 in mice in the control group and the 22B1 injection group; B shows HE sections of thyroid glands from mice in the control group, I under 100× microscope and II under 400× microscope; C shows HE sections of thyroid glands from mice in the 22B1 injection group, all under 400× microscope, with red arrows indicating columnar hyperplasia and black arrows indicating interstitial vascular congestion and dilation. Detailed Implementation

[0033] 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 should not be construed as limiting the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0034] The Sf9 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 first 289 amino acid residues of human TSHR were used as templates to amplify the TSHR289 construct, which was cloned into pFastBac1 and constructed by Shanghai Sangon Biotech Co., Ltd.; the adenovirus expressing the human TSHR A subunit was constructed by Shanghai Jikai Co., Ltd.; sequencing was performed by Shanghai Sangon Biotech Co., Ltd.; plasmid extraction kits were purchased from Qiagen; Lip2000 was purchased from Thermo Fisher Scientific; PEG1450 was purchased from Beijing Bio-Sens Biotechnology Co., Ltd.; Sf-900TMII SFM medium, 1640 medium, F12K medium, fetal bovine serum, HT and HAT 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 Yiqiao Shenzhou; affinity purification media was purchased from GE; fluorescence microscope was purchased from ZEISS; ELISA multi-well plate reader was purchased from Molecular Devices; ELISA plate washer was purchased from BIO-RAD; flow cytometer was purchased from BD; cAMP detection kit was purchased from R&D.

[0035] Example 1: Preparation of Monoclonal Antibody 22B1

[0036] 1. Expression of recombinant TSHR289 protein

[0037] Recombinant pFastBac containing the TSHR289 gene (expressing the first 289 amino acid residues of human TSHR) TM 1. Plasmid TOP10 E. coli were inoculated into LB medium and cultured overnight. The target plasmid was extracted and transformed into DH10 Bac E. coli, inoculated into SOC medium, and then inoculated into selection plates for overnight culture. White monoclonal colonies with good growth were selected, inoculated into SOC medium, and cultured overnight before being sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Colonies with correct sequencing were expanded, and plasmids were extracted. Recombinant Bacmid DNA was transfected into well-growing sf9 cells using Lip2000, and primary virus was obtained after one week. Cell supernatant was collected, centrifuged at 1000g for 5 min, and filtered through a 0.22µm filter to obtain the virus. The primary virus was used to further infect sf9 cells to expand the viral load. The virus was inoculated into High Five culture plates at a MOI of 5 pfu / cell. TMCells were infected, and the optimal time for protein harvesting was 72 hours post-infection. Western blot was used to detect the expression of the target protein TSHR289. Recombinant protein TSHR289 was purified by NI column affinity chromatography, and the purity of the recombinant protein TSHR289 was determined by SDS-Page assay.

[0038] 2. Mouse immunization

[0039] Six- to eight-week-old female BALB / c mice were immunized intramuscularly in the left and right femoral regions using adenovirus-expressed human TSHR A subunits. Immunization was repeated every three weeks for a total of three immunizations. One week after the second and third immunizations, serum titers were measured via tail vein blood collection. Mice with higher titers received the third and booster immunizations, respectively.

[0040] 3. Serum titer determination

[0041] Blood was collected from the tail vein at different time points. The blood was incubated at 37°C for 1 hour, then at 4°C for 2 hours, followed by centrifugation at 4000 rpm for 30 minutes to obtain serum. Serum was serially diluted from 1:500 to 1:160000. Purified TSHR289 recombinant protein was diluted to 5 μg / mL with PBS, and 100 μL / well was coated onto an ELISA plate and blocked overnight. 100 μL / well of serially diluted mouse serum was added (incubated at 37°C for 1 hour). The diluent was discarded, and the plate was washed 5 times with PBST. HRP-labeled anti-mouse IgG ELISA-labeled secondary antibody was added (incubated at 37°C for 1 hour). The diluent was discarded, and the plate was washed 5 times with PBST. A mixture of solutions A and B was added, and the plate was incubated at 37°C for 15 minutes. 50 μL of stop solution was added to each well, and the OD values ​​were read using an ELISA reader. 450 Numerical value.

[0042] 4. Preparation of hybridomas

[0043] Before fusion, peritoneal macrophages from 6-8 week old female BALB / c mice were used to prepare feeder cells. Spleen cells from mice with the highest antibody titers were fused with well-growing SP2 / 0 bone marrow tumor cells at a 10:1 ratio using PEG1450. After fusion, the fused cells were gently resuspended in HAT selection medium containing 20% ​​FBS, and 150 μL was added to each well of a 96-well cell culture plate containing feeder cells. The plates were incubated at 37°C in a 5% CO2 incubator, and cell growth was observed the following day. Two days after fusion, half the medium was replaced with HAT selection medium containing 20% ​​FBS. Cell fusion was observed under a microscope starting on the fourth day after fusion, and wells containing hybridoma cell clones were marked.

[0044] 5. Screening and clonal culture of positive hybridoma cells

[0045] Whole-plate ELISA was performed on the hybridoma cell culture supernatant. The first round of screening used indirect ELISA to select positive hybridoma cell clones. The primary antibody was the hybridoma cell culture supernatant, and the secondary antibody was an HRP-labeled anti-mouse secondary antibody. Cells with high absorbance OD were selected whenever possible. 450 Positive hybridoma cells with high readings, low clone numbers, and good cell condition were cultured for clonalization. Limiting dilution was used for 3-5 rounds of selection. In each round of cloning, positive monoclonal hybridoma cells were expanded and passaged. Indirect ELISA was performed during passage to assess cell passage stability, and cells were cryopreserved.

[0046] 6. Large-scale preparation and purification of monoclonal antibodies

[0047] Positive monoclonal hybridoma cells were transferred to 24-well cell culture plates, and then sequentially transferred to 6-well cell culture plates, T25, and T75 cell culture flasks for further expansion. When the cells in the culture flasks rapidly expanded, the cultured cells were collected, centrifuged at 1000 rpm for 5 min, and cultured at 1×10⁻⁶ cells / well. 6 Cells were injected intraperitoneally into pre-sensitized female BALB / c mice. One week later, ascites fluid was collected from the mice, centrifuged at 12000 rpm for 30 min, resuspended in PBS, and filtered through a 0.45 μm filter. Mouse hybridoma antibodies were purified using a Protein G pre-packed column. Protein concentration of the monoclonal antibody was determined using a GE NanoVue micro-spectrophotometer with the Protein A 280 method. The antibody was sterile filtered and stored at 2–8 °C. The anti-TSHR monoclonal antibody obtained through screening was designated 22B1; sequencing results showed that the amino acid sequences of its heavy chain variable region CDR1, CDR2, and CDR3 were as follows:

[0048] CDR1: GYTFTSYW (as shown in SEQ ID NO.1);

[0049] CDR2: IDPSDSET (as shown in SEQ ID NO.2);

[0050] CDR3: ARSGY (shown as SEQ ID NO.3);

[0051] The amino acid sequences of the four FR regions in the heavy chain variable region are as follows:

[0052] FR1: EVQLQQPGAELVRPGASVKLSCKAS (shown as SEQ ID NO.4);

[0053] FR2: MNWVKLRPGQGLEWIGL (shown in SEQ ID NO.5);

[0054] FR3: HYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYC (shown in SEQ ID NO. 6); FR4: WGQGTTLTVSS (shown in SEQ ID NO. 7);

[0055] The amino acid sequence of the heavy chain variable region is: EVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVK LRPGQGLEWIGLIDPSDSETHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSG YWGQGTTLTVSS (as shown in SEQ ID NO. 8);

[0056] Nucleotide sequence of the heavy chain variable region: GAGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTGGTGAG GCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGAACTGGGTGAAGCTGAGGCCTGGACAAGGCCTTGAATGGATTGGTTTGATTGATCCTTCAGACAGTGAAACTCACTACAATCAAATG TTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO.9).

[0057] The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are as follows:

[0058] CDR1: QSLLDSDGKTY (shown in SEQ ID NO.10);

[0059] CDR2: LVS (shown in SEQ ID NO.11);

[0060] CDR3: WQGTHFPHT (as shown in SEQ ID NO.12);

[0061] The amino acid sequences of the four FR regions in the light chain variable region are as follows:

[0062] FR1: SIQMTQTPLTLSVTIGQPASISCKSS (shown in SEQ ID NO.13);

[0063] FR2: LNWLLQRPGQSPKRLIY (shown in SEQ ID NO.14);

[0064] FR3: KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC (shown in SEQ ID NO. 15);

[0065] FR4: FGGGTKLEIKR (shown in SEQ ID NO.16);

[0066] The amino acid sequence of the light chain variable region is: SIQMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLL QRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPHTFG GGTKLEIKR (shown in SEQ ID NO. 17);

[0067] Nucleotide sequence of the light chain variable region: TCTATCCAGATGACACAGACTCCACTCACTTTGTCGGTT ACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCC TGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTCCTCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG(SEQ IDNO.18).

[0068] Example 2: Detection of properties of monoclonal antibody 22B1

[0069] 1. Antibody purity analysis

[0070] In this embodiment, anti-TSHR monoclonal antibody 22B1 was prepared in large quantities simultaneously using cell culture and mouse ascites methods. The cell culture supernatant and the crude extracted ascites were eluted with Protein G, and the protein concentration was measured to be 1000 μg / ml using a NanoVue micro spectrophotometer. Because the monoclonal antibody has a large molecular weight (mostly 150-180 kDa), it was subjected to β-mercaptoethanol reduction treatment. The purity of the purified monoclonal antibody was analyzed by reducing and non-reducing SDS-PAGE electrophoresis.

[0071] The results are as follows Figure 1 As shown in the figure, monoclonal antibody 22B1 was reduced by β-mercaptoethanol to 50 kDa (heavy chain) and 25 kDa (light chain). The molecular weight of the unreduced monoclonal antibody was approximately 150 kDa, consistent with the molecular weight of IgG. Electrophoresis results showed that high-purity monoclonal antibody was obtained using the Protein G affinity purification method.

[0072] 2. Antibody affinity detection

[0073] This embodiment uses surface plasmon resonance (SPR) analysis to detect the affinity kinetics of the anti-TSHR monoclonal antibody 22B1 and the TSHR289 fusion protein. A 10 mM sodium acetate solution (pH 4.5) was used as the coating buffer. The CM5 chip was activated under EDC / NHS conditions, and ethanolamine was used for blocking. The coating flow rate was 10 μL / min, and HBS-EP buffer was used as the running buffer. The purified TSHR289 fusion protein was coated onto the CM5 chip. Monoclonal antibody 22B1 was serially diluted to 3.0 μg / mL with HBS-EP buffer, and then sequentially flowed through the CM5 chip coated with purified TSHR289 at a flow rate of 30 μL / min. After binding, the protein was regenerated in Gly-HCl buffer (pH 1.5). Analysis was performed using Biacore T200 Evaluation Software 1.0 after the reaction.

[0074] The results are as follows Figure 2 As shown, affinity assays revealed that the purified TSHR289 protein had an affinity of 1.389 × 10⁻⁶ for the monoclonal antibody 22B1. 10 .

[0075] 3. Analysis of the binding affinity between the antibody and wild-type TSHR

[0076] This embodiment uses flow cytometry to detect the binding affinity of anti-TSHR monoclonal antibody 22B1 to wild-type human TSHR stably transfected in CHO cells. Stable CHO cells transfected with wild-type human TSHR and in good growth condition were digested with trypsin, cell counted, centrifuged at 1000 rpm for 5 min, and resuspended in 2% BSA. The binding affinity of the Fab fragment of monoclonal antibody 22B1 was detected under the same conditions after adding Fcblock. 1×10⁻⁶ cells were collected from both positive and untransfected cells. 6 Cells / tube were incubated with 10 μg of purified monoclonal antibody 22B1 at room temperature in the dark for 60 min. A control group was also incubated with an equal volume of PBS. Cells were washed three times with 2% BSA, then incubated with Per-CP-labeled secondary antibody at room temperature in the dark for 45 min. After washing three times with 2% BSA, the cells were resuspended and analyzed using a BD AccuriC6 flow cytometer. Results were analyzed using FlowJO software.

[0077] The results are as follows Figure 3 As shown, the binding rates of the IgG and Fab fragments of the anti-TSHR monoclonal antibody 22B1 to wild-type TSHR were 79.3% and 75.7%, respectively.

[0078] 4. Antibody activity analysis

[0079] This embodiment uses competitive ELISA to detect the cAMP production activity of CHO cells stably transfected with wild-type human TSHR stimulated by anti-TSHR monoclonal antibody 22B1. After digestion with trypsin, well-grown, stably transfected CHO cells were counted at 5 × 10⁻⁶ cells / cells. 4 Cells were evenly seeded per well in a 96-well cell culture plate and cultured in F12K complete medium for 24 hours. Cell adhesion was then observed. After good cell adhesion, the original medium was discarded, and the cells were cultured in serum-free medium for 2 hours. Then, purified monoclonal antibody 22B1, diluted at different concentrations and containing 1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine, was added to each well and incubated at 37°C for 4 hours. The cells were washed three times with PBS, and 150 μL / well of cell lysis buffer was added to the 96-well plate. The plate was then subjected to three freeze-thaw cycles at -80°C, and finally incubated overnight at -80°C. The following day, the cells were analyzed according to the ELISA kit manufacturer's method. This experiment was performed in triplicate, and the average value was used for statistical analysis.

[0080] The results are as follows Figure 4 As shown, the IgG of anti-TSHR monoclonal antibody 22B1 and the EC of the Fab fragment... 50 The concentrations were 0.26 μg / ml and 0.17 μg / ml, respectively.

[0081] 5. Analysis of antibody-mediated competitive binding to receptors with serum TRAb from GD patients

[0082] This embodiment uses competitive ELISA technology to detect the ability of TRAb in the serum of Graves' disease (GD) patients to competitively inhibit the binding of the anti-TSHR monoclonal antibody 22B1 to TSHR. Forty-six human serum samples were collected, including serum from 32 clinically diagnosed GD patients who were TRAb-positive and hyperthyroidism, and serum from 14 healthy individuals who were TRAb-negative (without any other diseases, including any autoimmune diseases). The serum samples were mixed with diluted HRP-labeled 22B1 monoclonal antibody and added to a commercially available ELISA plate coated with TSHR antigen. After incubation at 37°C for 60 min, the plate was washed five times with PBST, and a mixture of A+B solutions was added. After incubation at 37°C for 15 min, chromogenic solution C was added, and the plate was shaken well and then plotted on OD. 450 nm reading. The inhibition rate is calculated using the formula: 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.

[0083] The results are as follows Figure 5 The results show that, A: 22B1 was serially diluted (2000-fold, 4000-fold, and 8000-fold), and the binding ability of 22B1 to TSHR was detected by ELISA to determine the optimal dilution of 22B1; B: 22B1 was diluted at a ratio of 1:2000, and the competitive inhibition of TRAb in the serum of GD patients on the binding of 22B1 to TSHR was detected by ELISA. A small proportion of TRAb in the serum of GD patients could competitively inhibit the binding of TSHR on the monoclonal antibody 22B1 coated plate (with an inhibition rate of 50% as the standard for inhibition).

[0084] 6. Detection of antibody activity in vivo

[0085] In this embodiment, ELISA and HE section analysis were used to detect the bioactivity of anti-TSHR monoclonal antibody 22B1 in animals. Purified monoclonal antibody 22B1 was intraperitoneally injected at a dose of 250 μg into 6-8 week old female BALB / c mice twice a week for 3 weeks. The control group was injected with PBS in the same manner. Two days after the last injection, mice were anesthetized with isoflurane overdose, blood was collected, and thyroid tissue was obtained, fixed, dehydrated, embedded, sectioned, and stained with HE.

[0086] The results are as follows Figure 6 As shown, the serum TT4 level in 22B1-injected mice was (6.2±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). Figure 6(As shown in Figure A). However, mild hyperplasia of thyroid follicular epithelial cells was found in the HE section of mice injected with 22B1 (as shown in Figure A). Figure 6 As shown in I of B), interstitial vascular dilation and congestion ( Figure 6 (As shown in II of B).

[0087] The above activity analysis results show that:

[0088] This invention successfully prepared a monoclonal antibody 22B1 against TSHR289 protein. This monoclonal antibody 22B1 not only exhibits high affinity for purified TSHR289 protein but also binds to natural wild-type TSHR. Furthermore, monoclonal antibody 22B1 possesses thyroid-stimulating activity, stimulating cAMP production in CHO cells transfected with the full-length human TSHR receptor even at low concentrations (μg / ml). The thyroid-stimulating activity of the monoclonal antibody 22B1 described in this invention is similar to the reported activity of TRAb in human serum. Simultaneously, this invention also detected similar activity in the Fab fragment of monoclonal antibody 22B1.

[0089] This invention utilizes HRP-labeled monoclonal antibody 22B1. Competitive ELISA detection revealed that the binding of monoclonal antibody 22B1 to TSHR can be competitively inhibited by TRAb in the serum of GD patients. This indicates that monoclonal antibody 22B1 possesses human-like biological characteristics of TRAb, providing a theoretical basis and powerful tool for the current clinical detection of TRAb.

[0090] This provides a theoretical basis and a powerful tool for the current clinical detection of TRAb.

[0091] After injecting female BALB / c mice with the purified monoclonal antibody 22B1 of this invention, no significant change was observed in serum T4 levels compared to the control group. Simultaneously, mild proliferation of follicular epithelial cells and interstitial vasodilation were observed in the thyroid glands of the 22B1-injected mice, indicating that the monoclonal antibody 22B1 also exhibits certain thyroid-stimulating activity in vivo.

[0092] In summary, the monoclonal antibody 22B1 described in this invention exhibits high in vitro and in vivo thyroid-stimulating activity and can be competitively inhibited by TRAb from GD hyperthyroidism patients from binding to TSHR. Furthermore, compared to currently reported murine anti-TSHR289 monoclonal antibodies, ① the monoclonal antibody 22B1 described in this application has a high affinity for natural wild-type TSHR; ② it has strong thyroid-stimulating activity; and ③ it can be competitively inhibited by TRAb from GD hyperthyroidism patients from binding to TSHR, possessing the biological characteristics of a human-like TRAb. It has the potential to be used as a passive immunization agent product against TSHR monoclonal antibodies or for the development of in vitro diagnostic kits.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-thyrotropin receptor monoclonal antibody 22B1, characterized in that, The monoclonal antibody 22B1 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 22B1 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; and 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. 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. The nucleic acid encodes the antibody heavy chain and the antibody light chain of the monoclonal antibody 22B1 of any one of claims 1-3.

3. The monoclonal antibody 22B1 according to claim 1, characterized in that, 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.

4. A nucleic acid, characterized in that, 6. Use of the monoclonal antibody 22B1 of any one of claims 1-3 in the preparation of a reagent or kit for detecting TRAb.

5. The nucleic acid of claim 4, wherein, 7. Use of the monoclonal antibody 22B1 of any one of claims 1-3 in the preparation of a diagnostic kit for Graves' disease. ​ ​

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