Fully human TSH receptor blocking monoclonal antibody as well as preparation and application thereof

Abstract

The invention discloses a fully human TSH receptor (TSHR) blocking monoclonal antibody for treating hyperthyroidism as well as a preparation method and application of the fully human TSHR blocking monoclonal antibody. The method comprises the following steps: sorting plasma cells and memory single B cells for specifically recognizing TSHR in peripheral blood of a patient with high TSH-blocking antibody (TBAb) titer by using flow cytometry, cloning antibody light and heavy chains in vitro and performing recombinant expression, and performing antibody property screening verification by using hTSHR-CHO cells, so as to obtain the TSHR-TBAb-TSHR-TBAb. The blocking monoclonal antibody specifically targeting the human TSHR is obtained. The fully human TSH receptor blocking monoclonal antibody disclosed by the invention can be specifically combined with TSHR, has good inhibitory activity in in-vitro and in-vivo experiments, and has a wide application prospect in the aspect of treatment and development of Graves' diseases.

Description

Technical Field

[0001] The present invention belongs to the field of monoclonal antibody technology and humanized antibody drugs, and relates to a fully humanized TSH receptor blocking monoclonal antibody and its preparation and application. Background Art

[0002] Graves' disease (GD) is an organ-specific autoimmune disease caused by the combined effects of genetics and the environment, with increased secretion of thyroid hormones. It is the most common cause of hyperthyroidism. There are a large number of GD patients, with an incidence rate of about 0.2-2%. Like other autoimmune diseases, GD is more common in women of childbearing age, with a female incidence rate of about 5-10 times that of men. In recent years, with the change of living environment, the incidence of hyperthyroidism has been increasing. If the symptoms of hyperthyroidism are not controlled in time, it can affect the heart, leading to arrhythmia or heart failure, etc. Women of childbearing age may suffer from menstrual disorders, difficulty in pregnancy, miscarriage, etc. In addition, hyperthyroidism can mostly cause mental abnormalities such as tension, anxiety, irritability, etc., affecting the patient's study and life, and even causing mental illness in severe cases. At present, the treatment of GD mainly includes drugs, radioactive iodine and surgical treatment. The former has a long course of treatment, which is difficult for patients to adhere to, and the recurrence rate is high, with about 60-70% of patients recurring. Clinically, the treatment of GD has not changed substantially for many years, and it is still a choice between antithyroid drugs (ATD), radioactive iodine or surgery. Among them, antithyroid drugs have been used to treat Graves' disease for nearly 70 years. Except for the United States, doctors around the world regard ATD as the first choice for treating Graves' disease. However, after regular and systematic treatment, only some patients with hyperthyroidism can achieve cure, and the side effects are large. A considerable number of patients will relapse after a certain period of time. When the disease relapses, it worsens and generally requires radioactive iodine or surgical treatment, which greatly increases the economic and psychological burden on patients. In addition, radioactive iodine 131 nuclide treatment can easily lead to permanent hypothyroidism; the complications of surgical treatment cannot be ignored. The current treatment dilemma of GD means that there is an urgent need to find better alternative drugs in clinic.

[0003] TSH receptor antibodies (TRAb) are characteristic antibodies of GD. TRAb is a group of polyclonal antibodies that act on different binding sites of TSH receptors and can be divided into TSH receptor stimulating antibodies (TSH-stimulating antibody, TSAb), TSH receptor blocking antibodies (TSH-stimulating blocking antibody, TBAb) and neutral antibodies (neutral TSH receptor antibodies). Among them, TSAb binds to TSHR to produce a biological effect similar to TSH, which is the direct cause of Graves' disease. TBAb binds to TSH to block the binding of TSH to the receptor, inhibiting thyroid hyperplasia and thyroid hormone production. GD patients have both stimulating and blocking antibodies in their bodies, and the final outcome of their thyroid function depends on which antibody is dominant. This makes exogenous supplementation of a certain dose of blocking antibodies to bind to TSHR, thereby improving the pathophysiological effects of autoantibodies, becoming a new effective treatment for GD.

[0004] Humanized monoclonal antibodies can be divided into mouse humanized monoclonal antibodies and fully human monoclonal antibodies. Although mouse humanized monoclonal antibodies reduce the proportion of other non-human components to a certain extent, they still cannot completely eliminate all non-human components, and the affinity and original biological activity of the antibody will be reduced to a certain extent during the modification process. Fully humanized monoclonal antibodies directly amplify antibody genes from single human B cells, and obtain a large number of naturally paired antibody light and heavy chain genes by amplifying the antibody genes of isolated single plasma cells or memory B cells. Then, by expressing the paired antibody light and heavy chain genes, antibodies with antigen specificity and neutralizing activity are finally screened. This method has the advantages of rapidity, high throughput, and a small amount of cells required. The prepared fully human antibodies retain rich genetic diversity and natural pairing of light and heavy chain variable regions, which has great advantages. At present, the preparation of fully humanized antibodies against influenza, anthrax virus, and pneumococcus is based on this technology. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a fully human TSH receptor (TSHR) blocking monoclonal antibody for treating hyperthyroidism, and also provides the coding sequence of the antibody and a vector containing the coding sequence.

[0006] The present invention provides a fully human TSH receptor blocking monoclonal antibody, which binds to the TSH receptor and blocks the binding of TSH to the TSH receptor; the amino acid sequence of the antibody is shown in SEQ ID NOs: 1 and 2; the nucleotide sequence of the antibody is shown in SEQ ID NOs: 3 and 4.

[0007] Preferably, the amino acid sequence of the antibody has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 1, 2; and / or the nucleotide sequence of the antibody has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 3, 4.

[0008] Preferably, the amino acid sequence of the antibody is as shown in SEQ ID NO: 1, or has a sequence identity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more to SEQ ID NO: 2.

[0009] Preferably, the nucleotide sequence of the antibody is as shown in SEQ ID NO: 3, or has a sequence identity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more to SEQ ID NO: 4.

[0010] Preferably, the present invention provides an anti-human TSHR monoclonal antibody, named TBI80.

[0011] The antibody provided by the present invention binds to the TSH receptor to inhibit the signal transduction of the TSH receptor; inhibits the synthesis and secretion of thyroid hormones, and reduces hyperthyroidism caused by various reasons; after binding to the TSH receptor, it can significantly reduce the enlargement of the thyroid gland caused by hyperthyroidism and other reasons; and alleviates the inflammation, edema and hyperplasia of thyroid-related eye diseases.

[0012] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody in the preparation of drugs for treating hyperthyroidism, thyroid-related eye diseases and the like, in the preparation of drugs for inhibiting thyroid hyperplasia and / or thyroid hormone production, in the preparation of drugs for TSH receptor blocking, and in the preparation of drugs for antagonizing the activation effect of TSH on TSH receptors.

[0013] The present invention also provides a preparation, a medicine or a pharmaceutical composition, wherein the preparation, the medicine or the pharmaceutical composition comprises the above-mentioned fully human TSH receptor blocking monoclonal antibody.

[0014] The present invention also provides the use of the above-mentioned preparation, medicine or pharmaceutical composition in the preparation of medicines for treating hyperthyroidism and thyroid-related eye diseases, in the preparation of medicines for inhibiting thyroid hyperplasia and / or thyroid hormone production, in the preparation of medicines for blocking TSH receptors, and in the preparation of medicines for antagonizing the activation effect of TSH on TSH receptors.

[0015] The present invention also provides the use of the above-mentioned fully human TSH receptor blocking monoclonal antibody in the preparation of a preparation, a medicine or a pharmaceutical composition for detecting TSH receptor antibodies.

[0016] Furthermore, the preparation, medicine or pharmaceutical composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc.;

[0017] The preparation, medicine or pharmaceutical composition can be prepared into injection, sterile powder for injection, tablet, pill, capsule, lozenge, elixir, powder, granule, syrup, solution, tincture, aerosol, powder spray, suppository, etc. The preparations, medicines or pharmaceutical compositions in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0018] The preparation, drug or pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or wrapped with other substances.

[0019] The present invention also provides a reagent or a kit or a product, which contains the fully human TSH receptor blocking monoclonal antibody. Preferably, the reagent or kit for detecting hyperthyroidism and thyroid-related eye diseases contains the fully human TSH receptor blocking monoclonal antibody.

[0020] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody in the preparation of a reagent or a kit or a product for detecting hyperthyroidism and thyroid-related eye diseases.

[0021] The present invention also provides the use of the reagent, kit or product in preparing drugs for treating hyperthyroidism and thyroid-related eye diseases, in preparing drugs for inhibiting thyroid hyperplasia and / or thyroid hormone production, in preparing drugs for blocking TSH receptors, and in preparing drugs for antagonizing the activation effect of TSH on TSH receptors.

[0022] The present invention also provides a method for preparing a fully human TSH receptor blocking monoclonal antibody, which specifically comprises the following steps:

[0023] (1) Plasma cells and memory B cells targeting TSHR in the peripheral blood of patients with high TBAb activity were sorted, and single-cell RNA was extracted and cDNA was synthesized. The sorted single cells were amplified and verified by nested PCR for heavy chain (H), light chain (λ) and light chain (κ), and single-cell clones that were positive for both heavy chain and light chain were selected for subsequent cloning.

[0024] (2) Through nested PCR in vitro amplification, the BCR heavy and light chains of all single B cells were cloned into the heavy chain expression vector (AbVec-IGHG1), light chain expression vector (AbVec-hIgKappa) or light chain expression vector (AbVec-hIgLambda).

[0025] (3) After successfully obtaining the heavy chain and light chain recombinant plasmids, the candidate clones obtained are sequenced and compared to determine the number of nucleotide and amino acid sequences of the candidate antibodies obtained; the heavy chain and light chain expression plasmids are transfected to express monoclonal antibodies in vitro; the antigen binding ability and antibody blocking activity are further verified, and finally an antibody combination that can specifically target the target antigen, namely a fully human TSH receptor blocking monoclonal antibody, is obtained.

[0026] The preparation method of the present invention comprises the following steps: using flow cytometry to sort plasma cells and memory single B cells that specifically recognize TSHR in the peripheral blood of patients with high titers of TSH receptor blocking antibodies (TSH-stimulating blocking antibodies, TBAb), cloning antibody light and heavy chains in vitro and recombinantly expressing them, and using hTSHR-CHO cells to screen and verify antibody properties to obtain blocking monoclonal antibodies that specifically target human TSHR. Through the preparation method of the fully humanized TSH receptor blocking monoclonal antibody for treating hyperthyroidism proposed by the present invention, the target antibody sequence can be obtained in 3 weeks to 1 month.

[0027] Furthermore, the present invention verifies the properties of antibodies and evaluates their effects in vitro using hTSHR-CHO cells, and clarifies the blocking effects of antibodies through animal experiments.

[0028] The present invention also provides a nucleotide encoding the fully human TSH receptor blocking monoclonal antibody, the nucleotide sequence of which is one of the following sequences:

[0029] (1) the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 4;

[0030] (2) a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO:3 or SEQ ID NO:4;

[0031] (3) a nucleotide sequence after one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0032] (4) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (1), (2) or (3) above or its full-length complement; or

[0033] (5) A nucleotide sequence that is different from the nucleotide sequences of (1), (2), (3), and (4) above due to the degeneracy of the genetic code.

[0034] SEQ ID NO: 1 amino acid sequence

[0035] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu Arg LeuSer Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro Gly LysGly Leu Glu Trp Leu Ser Tyr Ile SerSer Ser Ala Ala Thr Val Phe Tyr Ala Asp Ser Val Gln Gly ArgLeu Thr Ile SerArg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAspThr Ala Val Tyr Tyr Cys Val Arg Glu Ala Val Ile Val Asp Gly Met Pro PheGlu Tyr Trp Gly GlnGly Ala Leu Val Thr Val Ser Ser

[0036] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSSAATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGALVTVSS)

[0037] SEQ ID NO:2

[0038] Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu SerPro Gly Glu Arg Ala Thr LeuSer Cys Arg Ala Ser Gln Ser Val Thr Ser Ser GlnLeu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala ProArg Leu Leu Ile Tyr Gly Arg Ser Gly Arg Ala ProArg Ala ProArg Ser Gly Arg Gly Ser GlyThr AspPhe Ala Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Cys Cys GlnGln TyrGly Thr Ser Ile Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[0039] (VHSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSQLAWYQQKPGQAPRLLIYGESSRATGIPDRFSGRGSGTDFALTISRLEPEDFAVYCCQQYGTSIAFGGGTKVEIK)

[0040] SEQ ID NO:3

[0041] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTC

[0042] CCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGT

[0043] CCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCA

[0044] ACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAA

[0045] GAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACT

[0046] GTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCC

[0047] CTGGTCACCGTCTCCTCAGC

[0048] SEQ ID NO: 4 base sequence

[0049] GTACATTCAGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGA

[0050] AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAGCAGCCAGTTAGCCTGGT

[0051] ACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGGTGAATCCAGCAGGGCC

[0052] ACTGGCATCCCAGACAGGTTCAGTGGCAGGGGTCTGGGACAGACTTCGCTCTCACCAT

[0053] CAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTGCTGTCAGCAGTATGGTACTTCAA

[0054] TCGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0055] The nucleotide sequence or at least part of the sequence described in the present invention can be expressed by a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include but are not limited to bacterial, insect cell and mammalian cell expression systems.

[0056] The present invention also provides an expression vector, which comprises the above-mentioned nucleotide encoding the fully human TSH receptor blocking monoclonal antibody.

[0057] The present invention also provides a host cell, comprising the above nucleotide and the above expression vector. Preferably, the host cell is a CHO-K1 cell or the like.

[0058] The method of the present invention, because there is no need to immunize animals, directly amplifies antibody genes from single human B cells, and amplifies the antibody genes of isolated single plasma cells or memory B cells to obtain a large number of naturally paired antibody light and heavy chain genes, and then expresses the paired antibody light and heavy chain genes to finally screen and obtain antibodies with antigen specificity and neutralizing activity. The method of the present invention has the advantages of rapidity, high throughput, and a small number of cells required. The prepared fully human antibodies retain rich genetic diversity and natural pairing of light and heavy chain variable regions, which has huge advantages. Compared with traditional hybridoma antibody preparation technology, the method of the present invention can significantly shorten the experimental cycle. Traditional hybridoma antibody preparation technology takes about 3 months to obtain antibody sequences, while the present invention only takes 3 weeks to 1 month to obtain antibody sequences, greatly reducing the workload and cost of antibody preparation.

[0059] The beneficial effects of the present invention also include: the fully human TSH receptor blocking monoclonal antibody of the present invention can specifically bind to TSHR, and has good inhibitory activity in in vitro and in vivo experiments, and has broad application prospects in the development of Graves' disease treatment. There are many GD patients worldwide, and the current treatment schemes for GD have many defects. The fully human TSHR blocking monoclonal antibody prepared by the present invention can not only effectively block the binding of TSH to the receptor, inhibit thyroid hyperplasia and / or thyroid hormone production, but also will not produce human anti-mouse antibody reaction, has little side effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0061] Figure 1The present invention uses flow cytometry to sort plasma cells and memory single B cells that specifically recognize TSHR from peripheral blood. Biotin-AF647 is used to label TSHR protein as bait, and other antibodies used for sorting are: CD19-PacificBlue, IgM-PE, CD27-BV605, CD38-PE-Cy7, and finally TSHR-specific plasma cells and memory single B cells are sorted as CD19+IgM-CD27+CD38-TSHR+.

[0062] Figure 2 This is a screening diagram for the antigen binding ability of the anti-human TSHR monoclonal antibody of the present invention.

[0063] Figure 3 This is a screening diagram for the blocking activity of the anti-human TSHR monoclonal antibody of the present invention. The results shown are mean ± standard deviation (n = 3).

[0064] Figure 4 The SDS-PAGE gel electrophoresis diagram of the in vitro expression of the TBI80 antibody of the present invention. R: reduced state; NR: non-reduced state.

[0065] Figure 5 This is a dilution blocking activity test diagram of the TBI80 antibody of the present invention in vitro, wherein the amount of bTSH added is 5 ng / ml, and the amount of hTSH added is 100 ng / ml.

[0066] Figure 6 The graph is a graph showing changes in T4 and TSH levels in rats injected intramuscularly with the TBI80 antibody of the present invention at different time points. The results shown are mean ± standard deviation (n = 5), *p < 0.05.

[0067] Figure 7 The effect of the Graves' disease mouse model treated three times with the TBI80 antibody of the present invention. A is the gross morphology of the thyroid gland after treatment; B is the weight of the thyroid gland; C is the change in T4 level in vivo; D is the gross morphology of the thyroid gland in situ in vivo; E is the morphology of the thyroid gland HE staining tissue, scale: 100 μm, the results shown are mean ± standard deviation (n = 5-6), *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0068] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0069] Example 1

[0070] Single cell sorting: Peripheral blood of volunteers with high TBAb titers was collected, and the immune density gradient centrifugation human B cell enrichment mixture (STEMCELL, catalog number: 15024) was added to the blood samples. -1077 (Sigma, Catalog No.: 10771) was used for density gradient centrifugation to obtain B cells in peripheral blood. CD19 + IgM - CD27 + CD38 - TSHR + Plasma cells and memory single B cells ( Figure 1 ).

[0071] Light and heavy chain variable region cloning: RNA bound to single cells was captured using the SPR1select nucleic acid fragment screening kit (Beckman Coulter, catalog number: B23317) and cloned according to SuperScript TM IV One-step RT-PCR System (Invitrogen, Catalog No.: 12594100) was used to synthesize cDNA. Using cDNA as a template, DreamTaq Green PCR 2X MasterMix (ThermoFisher, Catalog No.: K1081) was used to perform PCR amplification on the variable regions of the antibody light and heavy chains, respectively. The amplified PCR products were then subjected to agarose gel electrophoresis, and the bands with expected fragment sizes were cut and recovered. The DNA fragments were purified using the QIAquick Gel Extraction Kit (QIAGEN, Catalog No.: 28704) and sent for testing. The sequencing results were analyzed using the IgBLAST function of NCBI or the IMGT database, and the corresponding V and J gene cloning primers were selected for cloning PCR on the light and heavy chains. Subsequently, the HiFi DNA Assembly Master Mix (NEB, catalog number: E2621L) was used to connect the fragments, and the obtained light and heavy chain variable region sequences were cloned into the corresponding light and heavy chain expression vectors containing the light and heavy chain constant regions (NCBI GenBank numbers: FJ475055, FJ475056, FJ517647), respectively. The clones were selected after transformation plating, and the final sequences were determined by sequencing.

[0072] Blocking antibody screening:

[0073] 1. Antigen binding ability screening: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1 and incubated at 37°C and 5% CO 2After 3 days of culture, the culture supernatant was collected by centrifugation and the titer of TRAb antibody in the supernatant was detected according to the instructions of the human anti-thyrotropin receptor antibody enzyme-linked immunosorbent assay kit (Kelu, catalog number: ELK9540). The light and heavy chain combinations with binding ability lower than that of the blank control group were removed, and the remaining combinations were screened for blocking activity ( Figure 2 ).

[0074] 2. Blocking activity screening: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1 and incubated at 37°C with 5% CO 2 After 3 days of culture, the culture supernatant was collected by centrifugation. 100 μL of the supernatant was used to incubate hTSHR-CHO cells for 2 hours with 1 IU / L bTSH (Sigma). The cell lysate was collected to detect changes in cellular cAMP levels (R&D, Catalog No.: KGE002B). The antibody combination with strong blocking activity was retained for further verification ( Figure 3 ), among which K12 combination had the strongest inhibitory activity, and the corresponding monoclonal antibody was named TBI80, which was further expressed and purified.

[0075] Antibody expression and purification: The antibody light and heavy chain expression vectors were co-transfected into 293F cells at a ratio of 1:1; 37°C, 8% CO 2 After culturing with shaking at 130 rpm for 5 days, the culture supernatant was collected by centrifugation, filtered at 0.45 μm, and purified by Protein A (GenScript, Catalog No.: L00210) affinity chromatography to obtain antibody protein with high purity; the antibody concentration was determined by Bradford protein concentration method (Biyuntian, Catalog No.: P0006) and NanoDrop A280 method; the antibody expression was detected by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining (Biyuntian, Catalog No.: P0017F) Figure 4 ).

[0076] In vitro effect evaluation: The purified monoclonal antibody was diluted according to different concentration gradients, bTSH 5ng / ml or human TSH 100ng / ml was added, and hTSHR-CHO cells were incubated for 2 hours. The cell lysate was collected and the changes in cell cAMP levels were detected ( Figure 5 ) and found that TBI80 monoclonal antibody at a concentration of 1 μg / mL could effectively antagonize the activation of TSH on TSH receptors in a concentration-dependent manner.

[0077] In vivo effect evaluation: 6-8 week old male SD rats were selected, and each rat was intramuscularly injected with 0.1 mL TBI80 monoclonal antibody solution (200 μg TBI80 monoclonal antibody, buffer used: PBS; 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 8.1 mmol / L sodium dihydrogen phosphate, 1.5 mmol / L potassium dihydrogen phosphate, pH 7.4; containing 3% mannitol and 1% Tween-80). Blood was collected from the rat tail at 0 hour before injection, 4 hours, 24 hours, 48 ​​hours, and 72 hours after injection. The serum was separated by centrifugation at 4000 rpm for 5 minutes at 4 degrees and properly stored at -80°C for the determination of thyroxine T4 (Cloud Clone, catalog number: CEA452Ge) and thyrotropin TSH (Cloud Clone, catalog number: CEA463Ra). ( Figure 6 ), compared with 0 hours before injection, it was found that T4 decreased significantly 24 hours after injection, indicating that TBI80 still has inhibitory activity in the body. Later, due to the feedback increase of TSH, T4 level recovered.

[0078] A mouse model of Graves' disease was constructed. Adenovirus (Ad-TSHR 289) overexpressing the α subunit of human thyroid stimulating hormone receptor (TSHR) was injected intramuscularly into BALB / c mice three times with an interval of 3 weeks each time. The GD model was evaluated 3 weeks after the last immunization and divided into groups (control group, virus-untreated group, virus TBI80-treated group) for corresponding treatment. The treatment was repeated 3 weeks later, and blood was collected (to test thyroid function, liver and kidney function, etc.), and the size, weight and morphological changes of the thyroid gland were observed ( Figure 7 After three treatments with 50 μg TBI80, the T4 level decreased significantly compared with the untreated group, the thyroid size was basically restored, the weight and morphology were improved, the thyroid follicles were regular in morphology, and only a small number of epithelial cells still showed high columnar shape, which further demonstrated that the TSH receptor blocking monoclonal antibody TBI80 has in vivo inhibitory activity.

[0079] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A fully human TSH receptor blocking monoclonal antibody, characterized in that: The antibody binds to the TSH receptor and blocks the binding of TSH to the TSH receptor; the amino acid sequence of the antibody is shown in SEQ ID NOs: 1 and 2; the nucleotide sequence of the antibody is shown in SEQ ID NOs: 3 and 4.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the antibody has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 1, 2; and / or the nucleotide sequence of the antibody has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 3, 4.

3. A preparation, a medicine or a pharmaceutical composition, characterized in that: The preparation, medicament or pharmaceutical composition comprises the fully human TSH receptor blocking monoclonal antibody according to any one of claims 1-2.

4. Use of the fully human TSH receptor blocking monoclonal antibody as described in any one of claims 1 to 2, or the preparation, medicine or pharmaceutical composition as described in claim 3 in the preparation of a drug for treating hyperthyroidism and thyroid-related eye diseases, in the preparation of a drug for inhibiting thyroid hyperplasia and / or thyroid hormone production, in the preparation of a TSH receptor blocking drug, and in the preparation of a drug for antagonizing the activation effect of TSH on TSH receptors.

5. Use of the fully human TSH receptor blocking monoclonal antibody according to any one of claims 1 to 2 in the preparation of a preparation, a drug or a pharmaceutical composition for detecting TSH receptor antibodies.

6. A reagent or a kit, characterized in that: The reagent or kit contains the fully human TSH receptor blocking monoclonal antibody according to any one of claims 1-2.

7. Use of the reagent or kit as claimed in claim 6 in the preparation of a drug for treating hyperthyroidism or thyroid-related eye diseases, in the preparation of a drug for inhibiting thyroid hyperplasia and / or thyroid hormone production, in the preparation of a drug for blocking TSH receptors, or in the preparation of a drug for antagonizing the activation effect of TSH on TSH receptors.

8. Use of the fully human TSH receptor blocking monoclonal antibody according to any one of claims 1 to 2 in the preparation of a reagent or kit for detecting TSH receptor antibodies.

9. A nucleotide encoding the fully human TSH receptor blocking monoclonal antibody according to any one of claims 1 to 2, characterized in that: Its nucleotide sequence is one of the following: (1) the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (2) a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4; (3) a nucleotide sequence after one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (4) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (1), (2) or (3) above or its full-length complement; or (5) A nucleotide sequence that is different from the nucleotide sequences of (1), (2), (3), and (4) above due to the degeneracy of the genetic code.

10. An expression vector, characterized in that: It contains the nucleotide according to claim 9.

11. A host cell, characterized in that It comprises the nucleotide according to claim 9 and / or the expression vector according to claim 10.

12. A method for preparing a fully human TSH receptor blocking monoclonal antibody, characterized in that: The specific steps include: (1) Plasma cells and memory B cells targeting TSHR in the peripheral blood of patients with high TBAb activity were sorted, and single-cell RNA was extracted and cDNA was synthesized. The sorted single cells were amplified and verified by nested PCR for heavy chain H, light chain λ, and light chain κ. Single-cell clones that were positive for both heavy and light chains were selected for subsequent cloning; (2) The BCR heavy and light chains of all single B cells were cloned into the heavy chain expression vector AbVec-IGHG1, the λ light chain expression vector AbVec-hIgKappa, or the λ light chain expression vector AbVec-hIgLambda by nested PCR in vitro amplification; (3) After successfully obtaining the heavy chain and light chain recombinant plasmids, the obtained candidate clones are sequenced and compared and analyzed to determine the number of nucleotide and amino acid sequences of the obtained candidate antibodies; the heavy chain and light chain expression plasmids are transfected to express monoclonal antibodies in vitro; the antigen binding ability and antibody blocking activity are further verified, and finally an antibody combination that can specifically target the target antigen, namely the fully human TSH receptor blocking monoclonal antibody, is obtained.

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