Fully human TSH receptor-blocking monoclonal antibodies targeting CHK36 homolog cluster, preparation method therefor, and application thereof

Abstract

Provided are a set of fully human TSH receptor (TSHR)-blocking monoclonal antibodies targeting a CHK36 homolog cluster, a preparation method therefor, and an application thereof. The method comprises the following steps: using flow cytometry to sort plasma cells and single memory B cells that specifically recognize TSHR in peripheral blood of a patient with a high titer of TSH-blocking antibodies (TBAb), performing in vitro cloning of antibody light and heavy chains and performing recombinant expression, and using hTSHR-CHO cells to perform screening and validation of antibody properties to obtain a blocking monoclonal antibody that specifically targets human TSHR. The fully human TSH receptor-blocking monoclonal antibodies specifically bind to a TSHR, and effectively block signal transduction after a TSH binds to a receptor, inhibit the synthetic secretion of thyroid hormones, and significantly reduce and inhibit TSHR expression and fibrosis in orbital fibroblasts of effector cells associated with thyroid-related ocular diseases. The fully human TSH receptor-blocking monoclonal antibodies have broad application prospects in the treatment of Graves' disease (GD) and other diseases caused by hyperthyroidism, such as thyroid eye disease (TED).

Description

A group of CHK36 homologous cluster full human TSH receptor blocking monoclonal antibodies and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of monoclonal antibody technology and human antibody drugs, and relates to a group of full human TSH receptor blocking monoclonal antibodies and a preparation method and application thereof. BACKGROUND

[0002] The thyroid stimulating hormone receptor (TSHR) belongs to a seven-transmembrane G protein-coupled receptor and mainly exists on the cell membrane of thyroid follicular epithelial cells. The TSHR has a large extracellular domain, which is composed of a leucine-rich domain and a "hinge" region. Abnormal function of the TSHR can cause the occurrence of thyroid diseases. After thyroid stimulating hormone (TSH) binds to and activates the TSHR, downstream signal transduction can be mediated by the Gs and Gq / 11 pathways, which can regulate iodine transport absorption, iodine organicization, and the like, and participate in the synthesis and release of thyroid hormones, as well as the growth and differentiation of thyroid cells. On the other hand, the large extracellular segment of the TSHR can be divided into an alpha-subunit and a beta-subunit, and the alpha-subunit is prone to falling off. Under the background of genetic susceptibility, the TSHR peptide segment can act as an exposed autoantigen to induce the production of autoantibodies, i.e., thyroid stimulating hormone receptor antibodies (TRAb), in the body under the action of external environmental factors such as infection and mental trauma, and participate in the occurrence and development of autoimmune thyroid diseases (AITD), mainly including Graves' disease (GD) and Hashimoto's thyroiditis.

[0003] Graves' disease is an organ-specific autoimmune disease with increased secretion of thyroid hormone, which is caused by the combined action of genetics and environment, and is the most common cause of hyperthyroidism. The number of GD patients is large, and the incidence of the population is about 0.2-2%. Like other autoimmune diseases, GD is more common in women of childbearing age, and the incidence of women is about 5-10 times that of men. In recent years, with the change of living environment, the incidence of hyperthyroidism has been increasing, and if the symptoms of hyperthyroidism are not controlled in time, it can involve the heart, leading to arrhythmia or heart failure, etc., and can lead to menstrual disorders, difficulty in pregnancy, abortion, etc. in women of childbearing age. In addition, hyperthyroidism can mostly cause mental abnormalities such as tension, anxiety, restlessness, etc., affecting the learning and life of patients, 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, and patients are difficult to adhere to, and the recurrence rate is high, about 60-70% of patients will relapse. The treatment of GD in clinical practice has not changed substantially for many years, and it is still a choice between antithyroid drugs, radioactive iodine or surgery. Among them, the treatment of Graves' disease with antithyroid drugs has been nearly 70 years. Except for the United States, doctors all over the world regard ATD as the first choice for the treatment of Graves' disease. However, after regular and systematic treatment of hyperthyroidism patients, only part of the patients can achieve cure, and the side effects are large, and a considerable part of the patients will relapse after a certain period of time. When the disease relapses, the condition worsens, and generally radioactive iodine or surgical treatment is needed, which greatly increases the economic and psychological burden of patients. In addition, radioactive iodine 131 radionuclide treatment can easily lead to permanent hypothyroidism; the complications of surgical treatment cannot be ignored. The current treatment dilemma of GD means that it is urgent to find better alternative drugs in clinical practice.

[0004] TRAb is the general term for antibodies produced by the body against TSHR, and is a group of polyclonal antibodies, which can be divided into TSH receptor stimulating antibodies (TSH-stimulating antibody, TSAb), TSH receptor blocking antibodies (TSH-stimulating blocking antibody, TBAb) and neutral antibodies. Their recognition epitopes on TSHR are not the same, and the recognition epitopes of TSAb, TBAb and neutral antibodies are concentrated in the amino terminal (N terminal), carboxy terminal (C terminal) and hinge region of the extracellular region of TSHR. Among them, the thyroid stimulating antibody TSAb binds to the TSHR on the membrane of the thyroid follicular epithelial cells, produces a biological effect similar to TSH, causes hyperthyroidism, and is the direct cause of Graves' disease. TSAb stimulates G protein coupling and further activates adenylate cyclase (AC) to stimulate the production of cAMP, and the AC-cAMP pathway is in a state of continuous activity, stimulating thyroid cell proliferation, and the thyroid gland synthesizes and secretes excessive thyroid hormones, i.e. triiodothyronine (T3) and thyroxine (T4). TSH cannot normally play a feedback regulatory role on T3 and T4 due to competitive inhibition, resulting in continuous increase of T3 and T4, causing a series of reactions in the body, and further causing hyperfunction of thyroid cells. From the histological level, the thyroid of GD patients shows thickened and enlarged follicular cells. The gland shows typical T cell and B cell lymphocyte infiltration, and there is a characteristic of thyroiditis, with a small amount of cell apoptosis and a certain degree of follicular destruction. TBAb blocks the binding of TSH to the receptor and inhibits thyroid hyperplasia and thyroid hormone production. Stimulating and blocking antibodies coexist in GD patients, and the final result of thyroid function depends on which antibody is dominant. This makes it possible to supplement a certain dose of blocking antibodies to TSHR from the outside, thereby improving the pathophysiological effects of autoantibodies, and becomes a new effective scheme for treating GD.

[0005] Furthermore, TSH receptor blocking monoclonal antibodies can inhibit the synthesis and secretion of thyroid hormones by blocking the signal transduction after TSH binds to the receptor, thereby treating a series of diseases caused by hyperthyroidism, such as thyroid eye disease (thyroid eye disease, TED), also known as thyroid associated ophthalmopathy (Thyroid associated ophthalmopathy, TAO), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis and pretibial myxedema, etc.

[0006] Thyroid eye disease (TED) is a common orbital disease in adults, an autoimmune disease closely related to Graves' disease (GD), which can manifest as hyperemia and edema of the eyelid and conjunctiva, fibrosis and adiposis of the intraorbital tissue, causing eye movement disorders, strabismus and diplopia, which can cause blindness and disability, and is difficult to diagnose and treat, seriously affecting the quality of life of patients. China is one of the countries with a high incidence of GD in the world, and the prevalence of TED in GD patients of Asian descent is as high as 45%. The TED patient population is large, and their visual health and quality of life have been severely affected. As a difficult problem that has plagued the global medical scientific community for two centuries, TED currently lacks effective treatment options. Although there are various treatment options for TED, many patients do not respond well to existing drugs or cannot tolerate side effects. High-dose glucocorticoid pulse therapy can significantly reduce inflammation, but it has little effect on exophthalmos and diplopia, and long-term use has significant side effects, such as hypertension, diabetes, osteoporosis, and gastric ulcers. The effectiveness of glucocorticoids for some patients is only 50-75%. In addition, there is the drawback of short-term effectiveness, but relapse after discontinuation. Immunosuppressants can reduce eye inflammation by suppressing the overreaction of the immune system, but can cause serious side effects, such as increased risk of infection and liver and kidney function damage. Therefore, there is an urgent need for new, safe and effective drugs on the market.

[0007] Orbital fibroblasts (OF) are effector cells of TED autoimmune response, which have thyroid stimulating hormone receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R) on their surface. TSHR is ectopically highly expressed in CD34+ fibroblasts in the retrobulbar tissue of TED patients. TED immune response begins with abnormal recognition of autoantigen TSHR, and activated B cells produce TRAb. IGF-1R is another possible TED autoantigen, but it is not clear whether there are autoantibodies that directly stimulate IGF-1R. Studies have shown that IGF-1R and TSHR-mediated signal transduction pathways combine to cause the secretion of large amounts of hyaluronic acid (HAS). The antibody drug targeting IGF-1R (Tepezza) was approved by the FDA in 2020 for the treatment of TED. After treatment, exophthalmos and diplopia can be significantly improved, and nearly 40% of exophthalmos patients can basically return to normal. Although IGF-1R antibody drugs have significant efficacy, since IGF-1R is widely expressed in human organs, many significant side effects have been reported after large-scale clinical application, especially irreversible deafness and effects on the reproductive system. Because stimulating and blocking antibodies coexist in GD patients, the final result of thyroid function depends on which antibody is dominant. This makes it a new effective solution for treating thyroid eye disease to exogenously supplement a certain dose of blocking antibodies to inhibit TSHR.

[0008] Humanized monoclonal antibodies can be divided into murine humanized monoclonal antibodies and fully human monoclonal antibodies. Although the proportion of other non-human components is reduced to a certain extent in murine humanized monoclonal antibodies, all non-human components cannot be completely excluded, and the affinity and original biological activity of the antibodies are also reduced to a certain extent during the modification process. While fully human monoclonal antibodies directly amplify antibody genes from human single B cells, the antibody genes of single plasma cells or memory B cells separated by amplification are obtained in large quantities, and then the paired antibody light and heavy chain genes are expressed to ultimately screen antibodies with antigen specificity and neutralizing activity. This method has the advantages of rapidity, high throughput, and small amount of required cells, and the fully human antibodies prepared by this method retain rich genetic diversity and natural pairing of light and heavy chain variable regions, and have great advantages. At present, the preparation of fully human antibodies against influenza, anthrax virus and pneumococcus is based on this technology. SUMMARY

[0009] In view of the above prior art, the present application provides a group of fully human TSH receptor (TSHR) blocking monoclonal antibodies or antigen binding fragments thereof for treating hyperthyroidism, and also provides the coding sequence of the antibodies and the vectors containing the coding sequence.

[0010] In the present application, the term "TSHR" refers to a full-length human thyroid-stimulating hormone receptor having an amino acid sequence as shown in SEQ ID NO: 25, or a variant or fragment highly homologous to the thyroid-stimulating hormone receptor. Preferably, such variant and fragment have 70-99.9% homology with the amino acid sequence as shown in SEQ ID NO: 25.

[0011] The present application provides a group of fully human TSH receptor blocking monoclonal antibodies or antigen binding fragments thereof, which bind to TSH receptor and block the binding of TSH to TSH receptor; the fully human TSH receptor blocking monoclonal antibodies or antigen binding fragments thereof comprise a heavy chain variable region and a light chain variable region.

[0012] The heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, and the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0013] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 39.

[0014] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 39.

[0015] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 40.

[0016] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41.

[0017] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 32, SEQ ID NO: 37, SEQ ID NO: 42.

[0018] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 43.

[0019] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 44.

[0020] The amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 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% sequence identity with one of SEQ ID NO: 1, 3, 5, 7, 9, 11.

[0021] The amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 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% sequence identity with one of SEQ ID NO: 2, 4, 6, 8, 10, 12.

[0022] The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), Fav, dsFv, sc(Fv)2, single domain antibody (dAb).

[0023] In the present application, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof are as shown in SEQ ID NO: 13-SEQ ID NO: 24.

[0024] Preferably, the nucleotide sequence encoding the heavy chain variable region and the light chain variable region of said fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof 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 to SEQ ID NO: 13- SEQ ID NO: 24.

[0025] SEQ ID NO: 1 (CHK8_1 amino acid sequence)

[0026] SEQ ID NO: 2 (CHK8_2 amino acid sequence)

[0027] SEQ ID NO: 13 (CHK8_3 nucleotide sequence)

[0028] SEQ ID NO: 14 (CHK8_4 nucleotide sequence)

[0029] SEQ ID NO: 3 (CHK9_1 amino acid sequence)

[0030] SEQ ID NO: 4 (CHK9_2 amino acid sequence)

[0031] SEQ ID NO: 15 (CHK9_3 nucleotide sequence)

[0032] SEQ ID NO: 16 (CHK9_4 nucleotide sequence)

[0033] SEQ ID NO: 5 (CHK11_1 amino acid sequence)

[0034] SEQ ID NO: 6 (CHK11_2 amino acid sequence)

[0035] SEQ ID NO: 17 (CHK11_3 nucleotide sequence)

[0036] SEQ ID NO: 18 (CHK11_4 nucleotide sequence)

[0037] SEQ ID NO: 7 (CHK14_1 amino acid sequence)

[0038] SEQ ID NO: 8 (CHK14_2 amino acid sequence)

[0039] SEQ ID NO: 19 (CHK14_3 nucleotide sequence)

[0040] SEQ ID NO: 20 (CHK14_4 nucleotide sequence)

[0041] SEQ ID NO: 9 (CHK20_1 amino acid sequence)

[0042] SEQ ID NO: 10 (CHK20_2 amino acid sequence)

[0043] SEQ ID NO: 21 (CHK20_3 nucleotide sequence)

[0044] SEQ ID NO: 22 (CHK20_4 nucleotide sequence)

[0045] SEQ ID NO: 11 (CHK36_1 amino acid sequence)

[0046] SEQ ID NO: 12 (CHK36_2 amino acid sequence)

[0047] SEQ ID NO: 23 (CHK36_3 nucleotide sequence)

[0048] SEQ ID NO: 24 (CHK36_4 nucleotide sequence)

[0049] SEQ ID NO: 25 (human TSHR amino acid sequence)

[0050] Table 1: CDR regions of heavy (H) and light (L) chains of CHK36 homocluster fully human TSH receptor blocking monoclonal antibodies

[0051] This invention comprises a group of fully human TSH receptor blocking monoclonal antibodies or their antigen-binding fragments, namely CHK36, CHK9, CHK8, CHK14, CHK20, and CHK11. These antibodies share high homology with CHK36, and are therefore named a group of CHK36 homology cluster fully human TSH receptor blocking monoclonal antibodies or their antigen-binding fragments. In the table above, k1-70 represent a previously reported TSH receptor blocking monoclonal antibody with blocking activity. In comparison, the sequences provided in this invention are significantly different and show no homology whatsoever.

[0052] Preferably, the present invention proposes a set of fully human TSH receptor blocking monoclonal antibodies or their antigen-binding fragments, named CHK36 homology cluster fully human TSH receptor blocking monoclonal antibodies or their antigen-binding fragments.

[0053] In this invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment is a TSH antagonist.

[0054] In this invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment is an antagonist of thyroid-stimulating antibodies.

[0055] In this invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment each contains a V H Region (heavy chain variable region), the V H The region contains complementarity-determining regions (CDRs) of amino acid sequences as shown in SEQ ID NO: 26-SEQ ID NO: 28 (see Table 1).

[0056] In this invention, each antibody contains a V L Region (light chain variable region), the V L The region contains CDRs of amino acid sequences as shown in SEQ ID NO: 29, 35, 39 (see Table 1), CDRs of amino acid sequences as shown in SEQ ID NO: 30, 35, 40 (see Table 1), CDRs of amino acid sequences as shown in SEQ ID NO: 31, 36, 41 (see Table 1), CDRs of amino acid sequences as shown in SEQ ID NO: 32, 37, 42 (see Table 1), CDRs of amino acid sequences as shown in SEQ ID NO: 33, 38, 43 (see Table 1), or CDRs of amino acid sequences as shown in SEQ ID NO: 34, 35, 44 (see Table 1).

[0057] In this invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment contains one or more amino acid sequences that are substantially homologous to these CDRs.

[0058] The full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof provided by the present application can inhibit the signal transduction of the TSH receptor after binding to the TSH receptor, inhibit the synthesis and secretion of thyroid hormone, reduce hyperthyroidism caused by various reasons, and significantly reduce the goiter caused by hyperthyroidism and the like after binding to the TSH receptor, and reduce inflammation, edema and hyperplasia and the like of thyroid associated ophthalmopathy.

[0059] The thyroid associated ophthalmopathy (TAO) provided by the present application is also called thyroid eye disease (TED).

[0060] The present application also provides a preparation, a drug or a pharmaceutical composition comprising the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0061] The present application also provides a reagent or kit containing the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0062] The present application also provides a nucleotide encoding the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above, and the nucleotide sequence is one of the following sequences:

[0063] (a) the nucleotide sequence encoding the heavy chain variable region is as shown in one of SEQ ID NO: 13, 15, 17, 19, 21 and 23; and the nucleotide sequence encoding the light chain variable region is as shown in one of SEQ ID NO: 14, 16, 18, 20, 22 and 24;

[0064] (b) 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 to SEQ ID NO: 13-SEQ ID NO: 24;

[0065] (c) a nucleotide sequence obtained by adding, substituting, deleting or inserting one or several nucleotides to the nucleotide sequence as shown in SEQ ID NO: 13-SEQ ID NO: 24;

[0066] (d) a nucleotide sequence which hybridizes to the nucleotide sequence of (a), (b) or (c) above or its full-length complement under stringent conditions; or,

[0067] (e) a nucleotide sequence that differs from the aforementioned (a), (b), (c), (d) due to the degeneracy of the genetic code;

[0068] wherein the nucleotide sequence of the nucleotide or a part thereof encodes an antibody V H domain; and an antibody V L domain or is selected from the CDRs as shown in SEQ ID NO: 26-SEQ ID NO: 44 (see Table 1).

[0069] The present application also provides a vector containing the nucleotide as described above.

[0070] The present application also provides a host cell comprising the nucleotide as described above, and / or the vector as described above.

[0071] The present application also provides a cell which is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above, and / or the nucleotide as described above, and / or the vector as described above.

[0072] The present application also provides a cell which is an isolated cell expressing the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0073] The present application also provides a cell which is an isolated cell secreting the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0074] The present application also provides a composition comprising a determined concentration of TSH receptor autoantibodies and comprising a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0075] The present application also provides a pharmaceutical composition for administration to a mammalian subject for the treatment of a thyroid-related condition, comprising a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above and a pharmaceutically acceptable carrier;

[0076] wherein the thyroid-related condition is selected from the group consisting of hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema.

[0077] Further, the formulation, medicament or pharmaceutical composition can further comprise a physiologically compatible excipient, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorptive carriers, lubricants, etc.

[0078] Further, the formulation, medicament or pharmaceutical composition can be prepared as an injection, a sterile powder for injection, a tablet, a pill, a capsule, a lozenge, a tincture, a powder, a granule, a syrup, a solution, a tincture, an aerosol, a powder mist, or a suppository, etc. The formulation, medicament or pharmaceutical composition of the above-mentioned various dosage forms can be prepared according to the conventional methods in the field of pharmacy.

[0079] Further, the formulation, medicament or pharmaceutical composition can be introduced into the body, such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue, by injection, spraying, nose drops, eye drops, penetration, absorption, physical or chemical mediated methods; or mixed or wrapped with other substances before being introduced into the body.

[0080] Preferably, the reagent or kit for detecting hyperthyroidism and thyroid-related eye disease contains the full human TSH receptor blocking monoclonal antibody.

[0081] Further, the nucleotide sequence or at least part of the sequence 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.

[0082] Further, the vector can be a plasmid, a virus or their fragments, as well as various types of vectors known to those skilled in the art.

[0083] Preferably, the host cell is a CHO-K1 cell or the like.

[0084] Specifically, the composition can comprise a determined concentration of TSHR autoantibodies having TSH antagonist activity, and includes a full human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to the present application.

[0085] Further, the pharmaceutical composition is suitable for administration to humans; preferably, the pharmaceutical composition according to the present application has no significant adverse effects on the immune system of the subject.

[0086] Further, the pharmaceutical composition comprises one or more additional thyroid-stimulating hormone receptor antagonists.

[0087] Further, the pharmaceutical composition is used for treating a thyroid-related condition in an injectable form.

[0088] Preferably, said pharmaceutical composition for the treatment of Graves' ophthalmopathy is in the form of an intravenous injection formulation or an eye drop.

[0089] Preferably, said pharmaceutical composition for the treatment of pretibial myxedema is in the form of a topical administration.

[0090] In particular, said pharmaceutical composition comprises any antibody according to the present application and any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of this application include, but are not limited to, buffers such as phosphate, glycine, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), sorbitan, potatium sorbate, water, sodium chloride, salt or electrolyte such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, partially

[0091] The pharmaceutical composition can be formulated in a wide variety of dosage forms. Such formulations include, but are not limited to, capsules, tablets, aqueous suspensions, solutions, rectal suppositories, enemas, ointments, lotions, creams, nasal sprays, inhalers. Preferably, the formulation is a solution or an ointment.

[0092] In particular, the pharmaceutical composition can be in the form of a sterile injectable preparation, such as a sterile injectable oil or water suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, mannitol, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables. The sterile oil solutions or suspensions can also contain long chain alcohol diluents or dispersants.

[0093] In particular, the pharmaceutical composition can be administered topically, by spray inhalation, orally, parenterally, by eye drop or eye ointment, buccal, vaginal, rectal, nasal administration or via an implanted reservoir, and the like. Preferably, the administration is oral or by injection. The term "parenterally" as used herein includes subcutaneous, intracutaneous, intra-articular, intrasynovial, intrasternal, intravenous, intramuscular, intralesional, intracranial, intraarterial, and intrathecal injection or infusion techniques.

[0094] In particular, the pharmaceutical compositions can be administered in a form suitable for rectal administration. Such compositions will be in the form of suppositories for example containing the active compound in a mixture with a non-irritating excipient. Examples of excipients include, but are not limited to, cocoa butter and other glycerides.

[0095] In particular, the pharmaceutical compositions can be orally administered in any orally acceptable dosage form including, but not limited to, tablets, capsules, aqueous suspensions and solutions. In the case of tablets, commonly used carriers include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For

[0096] In particular, the pharmaceutical compositions can be administered in the form of a nasal spray or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0097] In particular, the pharmaceutical compositions of the present application are particularly useful for topical administration when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active compound suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this application include, but are not limited to, mineral oil, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable cream or lotion containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, and water. The pharmaceutical compositions of this application can also be formulated to be suitable for rectal administration or for administration to the lower intestinal tract by means of suppositories or suitable enemas. The present application also includes topical transdermal patches.

[0098] The present application also provides a method of producing a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof, which method comprises the steps of:

[0099] The first step is to sort the TSHR-aimed plasma cells and memory B cells in the peripheral blood of patients with high TBAb activity, to carry out single cell RNA extraction and cDNA synthesis, and to verify the sorted single cells by heavy chain H, light chain lambda and light chain kappa amplification through nested PCR, and to select single cells with positive heavy chain and light chain for subsequent cloning;

[0100] The second step is to amplify and clone the BCR heavy chain and light chain of all single B cells into the expression heavy chain vector AbVec-IGHG1, the lambda light chain expression vector AbVec-hIgKappa or the lambda light chain expression vector AbVec-hIgLambda through nested PCR in vitro.

[0101] The third step is to sequence and compare the obtained candidate clones after successfully obtaining the heavy chain and light chain recombinant plasmids, to determine the number of nucleotide and amino acid sequences of the obtained candidate antibodies, to transfect the heavy chain and light chain expression plasmids, to express the monoclonal antibodies in vitro, to further verify the antigen binding capacity and antibody blocking activity, and to finally obtain an antibody combination that can be specifically aimed at a target antigen, i.e., the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof.

[0102] The application also provides a method for treating a thyroid-related disease in a mammalian subject or in a cell derived from the subject, which comprises contacting the subject or the cell with the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0103] The thyroid-related disease is selected from hyperthyroidism, thyroid-related eye disease (Graves' eye disease), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis and pretibial myxedema, etc.

[0104] The application also provides a method for inhibiting the stimulation of TSH receptors by thyroid stimulating antibodies in the thyroid of a mammalian subject, which comprises contacting the subject with the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0105] Preferably, the binding of thyroid stimulating antibodies to TSH receptors is prevented.

[0106] The application also provides a method for inhibiting the binding of thyroid stimulating autoantibodies to TSH receptors outside the thyroid in a mammalian subject, which comprises contacting the subject with the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0107] In particular, the ectopic TSH receptor is located in retro-orbital tissue and / or pre-tibial tissue of the subject;

[0108] Preferably, the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof can block the binding of TSH receptor autoantibodies to ectopic TSH receptors.

[0109] The application also provides a method for treating thyroid cancer or metastatic thyroid cancer in the thyroid of a subject or in a thyroid cell derived from a subject, said method comprising contacting said cancer cell with a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above, with the aim of inhibiting constitutive thyroid stimulating hormone receptor activity in said cell;

[0110] Preferably, the regrowth of the thyroid cancer cell is prevented or delayed.

[0111] The application also provides a method for treating hyperthyroidism caused by constitutive thyroid activity in a subject or in a thyroid cell derived from a subject, said method comprising contacting said subject or cell with a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above, with the aim of inhibiting such hyperthyroidism.

[0112] The application also provides a method for identifying a molecule capable of inhibiting the binding of thyroid stimulating antibodies to TSH receptors, said method comprising providing at least one fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above as a reference;

[0113] Preferably, a molecule is selected which prevents the binding of thyroid stimulating antibodies to TSH receptors.

[0114] The application also provides a method for identifying a molecule capable of inhibiting the binding of thyroid blocking antibodies to TSH receptors, said method comprising providing at least one fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above as a reference;

[0115] Preferably, a molecule is selected which prevents the binding of thyroid blocking antibodies to TSH receptors.

[0116] In particular, the thyroid-related disorder is selected from hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis and pre-tibial myxedema, among others.

[0117] Preferably, the subject treated in the above method is a human.

[0118] In the present application, the preparation method comprises the following steps: using flow cytometry to sort the plasma cells and memory single B cells specifically recognizing TSHR in peripheral blood of patients with high TSH-stimulating blocking antibody (TBAb) titer, cloning the antibody light and heavy chains in vitro and recombinantly expressing, and using hTSHR-CHO cells to screen and verify the antibody properties, so as to obtain blocking monoclonal antibodies specifically targeting human TSHR. Through the preparation method of the blocking monoclonal antibodies of human TSHR provided in the present application, the target antibody sequence can be obtained in 3 weeks to 1 month.

[0119] Further, the present application verifies and evaluates the antibody properties and effects in vitro by using hTSHR-CHO cells.

[0120] The present application also provides the use of the human TSHR blocking monoclonal antibody or antigen binding fragment thereof as described above for treating a thyroid-related disease.

[0121] The present application also provides the use of the human TSHR blocking monoclonal antibody or antigen binding fragment thereof as described above for the preparation of a medicament for treating a thyroid-related disease.

[0122] The present application also provides the use of the human TSHR blocking monoclonal antibody or antigen binding fragment thereof as described above for the preparation of a reagent or kit for detecting TSHR antibodies.

[0123] The present application also provides the use of the human TSHR blocking monoclonal antibody or antigen binding fragment thereof as described above for the preparation of a reagent or kit or product for detecting hyperthyroidism and thyroid-related eye disease.

[0124] The present application also provides the use of the human TSHR blocking monoclonal antibody or antigen binding fragment thereof as described above, or the substance as described above, or the method as described above for the preparation of a preparation, medicament or pharmaceutical composition for detecting TSHR antibodies, a medicament for treating hyperthyroidism and thyroid-related eye disease, a medicament for inhibiting thyroid hyperplasia and / or thyroid hormone production, a TSHR blocking medicament, and a medicament for antagonizing the activation of TSH on TSHR.

[0125] Specifically, the thyroid-related disease in the above method is selected from hyperthyroidism, thyroid-related eye disease (Graves' eye disease), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema, etc.

[0126] The method of the present application does not need to immunize animals, and a large number of naturally paired antibody light and heavy chain genes are obtained by directly amplifying antibody genes from human single B cells, by amplifying the antibody genes of the isolated single plasma cells or memory B cells, and then by expressing the paired antibody light and heavy chain genes, and finally screening to obtain antibodies with antigen specificity and neutralizing activity. The method of the present application has the advantages of rapidity, high throughput, and small amount of required cells, and the prepared fully human antibodies retain rich genetic diversity and natural pairing of light and heavy chain variable regions, and have great advantages. Compared with the traditional hybridoma antibody preparation technology, the experimental period can be significantly shortened, and the traditional hybridoma antibody preparation technology generally needs about 3 months to obtain antibody sequences, while the present application only needs 3 weeks to 1 month to obtain antibody sequences, greatly reducing the workload and cost of antibody preparation.

[0127] The present application also has the advantages that the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof can effectively block the signal transduction after TSH binds to the receptor, inhibit the synthesis and secretion of thyroid hormones, and significantly inhibit the fibrosis of effector cells of thyroid-related eye disease, i.e. orbital fibroblasts. It can be used for treating a series of diseases caused by hyperthyroidism, such as thyroid eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0128] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0129] Figure 1 is a flow cytometry technology for sorting TSHR-specific plasma cells and memory single B cells from peripheral blood. Biotin-AF647 is used to label TSHR protein as a decoy, and other antibodies used for sorting are: CD19-Pacific Blue, 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 + .

[0130] Figure 2 is a diagram of the screening of the blocking activity of the anti-human TSHR monoclonal antibodies of the present application. The results shown are the mean ± standard deviation (n = 3). The dotted line value is 30%.

[0131] Figure 3 is a diagram of the results of the phylogenetic tree analysis of the sequences of the anti-human TSHR monoclonal antibodies of the present application. The percentage of replicate trees in which a given clade is found is shown next to the name of the clade in the branch.

[0132] Figure 4 is an evaluation of the inhibitory effect of CHK36 antibody on orbital fibroblasts in the present application. The horizontal line number indicates the statistical difference (P value) of the antibody group compared with the control group (Control), and a value less than or equal to 0.05 is considered to be significantly different. DETAILED DESCRIPTION

[0133] The present application is further described in conjunction with the following specific examples and accompanying drawings. The processes, conditions, experimental methods and the like used herein are those conventionally known in the art, unless specifically noted otherwise in the present application.

[0134] The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the application or its application or uses. All other embodiments obtained by those of ordinary skill in the art by applying the examples in the present application without creative work are within the scope of protection of the present application.

[0135] In all examples shown and discussed herein, any specific value should be interpreted as merely exemplary, and not as a limitation. Thus, other examples of the exemplary embodiments can have different values.

[0136] The present application proposes a group of therapeutic hyperthyroidism fully human TSH receptor (TSHR) blocking monoclonal antibodies and a preparation method and application thereof. The method described in the present application comprises the following steps: using flow cytometry to sort the specific TSHR-recognizing plasma cells and memory single B cells in the peripheral blood of patients with high TSH-stimulating blocking antibody (TBAb) titer, cloning the antibody light and heavy chains in vitro and recombinantly expressing them, and using hTSHR-CHO cells to screen and verify the antibody properties, to obtain blocking monoclonal antibodies that specifically target human TSHR. The fully human TSH receptor blocking monoclonal antibodies described in the present application can specifically bind to TSHR and have good inhibitory activity in in vitro and in vivo experiments, and have broad application prospects in the development of Graves' disease treatment.

[0137] The test materials used in the examples are conventional biochemical reagents unless otherwise specified.

[0138] Example 1

[0139] Single cell sorting: Peripheral blood of volunteers with high TBAb titers was collected, and B cell enrichment cocktail (STEMCELL, Cat# 15024) was added to the blood sample after Ficoll-Paque PLUS (Sigma, Cat# 10771) density gradient centrifugation to obtain B cells from peripheral blood. CD19 + IgM - CD27 + CD38 - TSHR + plasma cells and memory single B cells (Figure 1).

[0140] Light and heavy chain variable region cloning: RNA bound to single cells was captured using SPRlselect Nucleic Acid Fragment Screening Kit (Beckman Coulter, Cat# B23317), and cDNA was synthesized according to the instruction of Superscript TM IV One-Step RT-PCR System (Invitrogen, Cat# 12594100). Using cDNA as template, DreamTaq Green PCR 2X Master Mix (ThermoFisher, Cat# K1081) was used to amplify the antibody light and heavy chain variable regions respectively. The amplified PCR products were then detected by agarose gel electrophoresis, and the bands with expected fragment sizes were cut and recovered, and the DNA fragments were purified using QIAquick Gel Extraction Kit (QIAGEN, Cat# 28704) and sent for sequencing. The sequencing results were analyzed by IgBLAST function of NCBI or IMGT database, and the corresponding V and J gene cloning primers were selected for light and heavy chain cloning PCR. Subsequently, HiFi DNA Assembly Master Mix (NEB, Cat# E2621L) was used for fragment ligation, and the obtained light and heavy chain variable region sequences were cloned into the corresponding light and heavy chain expression vectors (NCBI GenBank number: FJ475055, FJ475056, FJ517647) containing light and heavy chain constant regions, respectively. The transformed plates were cloned and the final sequences were determined by sequencing.

[0141] Blocking antibody screening:

[0142] ​​1. Screening of antigen binding ability: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1. After 3 days of culture at 37°C and 5% CO2, 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, item number: ELK9540). The light and heavy chain combinations with a binding ability lower than the blank control group were removed, and the remaining combinations were subjected to blocking activity screening.

[0143] 2. Screening of blocking activity: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1. After 3 days of culture at 37°C and 5% CO2, the culture supernatant was collected by centrifugation, and 100 μL of the supernatant was used to incubate hTSHR-CHO cells for 2 hours with the addition of 1 IU / L bTSH (Sigma). Then, the cell lysate was collected, and the change in cAMP level was detected (R&D, item number: KGE002B).

[0144] Antibody expression and purification: The antibody light and heavy chain expression vectors were co-transfected into 293F cells at a ratio of 1:1. After 5 days of culture at 37°C and 8% CO2 with 130 rpm shaking, the culture supernatant was collected by centrifugation, filtered with 0.45 μm, and purified by Protein A (Jinse, item number: L00210) affinity chromatography to obtain antibody proteins with high purity. The antibody concentration was determined by Bradford protein concentration method (Biyun Tian, item number: P0006) and NanoDrop A280 method.

[0145] Evaluation of in vitro TSHR inhibition effect: The purified monoclonal antibodies were diluted according to different concentration gradients, and 5 ng / ml of bTSH was added for co-incubation of hTSHR-CHO cells for 2 hours. Then, the cell lysate was collected, and the change in cAMP level was detected. If the inhibition percentage of cAMP generation was greater than 30%, the monoclonal antibody was considered to have inhibitory activity. It was found that the monoclonal antibody at a concentration of 1 μg / mL could effectively antagonize the activation of TSH on TSH receptor and showed a concentration-dependent effect. The inhibition percentage of cAMP generation of CHK36 at a concentration of 1 μg / mL was 94%, and the inhibition activity at a concentration of 2 μg / mL was as high as 92%.

[0146] Phylogenetic analysis: The phylogenetic analysis of antibodies was performed using MEGA 11 analysis software, and the phylogenetic tree was constructed using the neighbor-joining method. The reliability of the phylogenetic tree was evaluated using the Bootstrap value. The percentage of replicate trees in which the relevant taxa clustered together in the bootstrap test (1000 replicates) is shown below the branches. The evolutionary distance was calculated using the Poisson correction method. Figure 3 is a rootless optimal tree combined with the results of the in vitro inhibition activity assay. Antibody K1-70 is a reported inhibitory antibody, and the rest are antibodies obtained by screening. The closer the evolutionary distance of the antibodies, the closer the inhibition activity. In the group of THSR inhibitory monoclonal antibodies screened in the present application, CHK36 and CHK9 have a very close evolutionary distance, and they have similar inhibitory efficiency on TSHR function. They are the two human monoclonal antibodies with the strongest inhibitory activity in this group (the inhibitory activity is greater than 90% at 2 μg / mL). Further analysis results show that K1-70 has a distant evolutionary distance from CHK36, the antibody with the strongest inhibitory activity in the present application, indicating that the sequences of the two antibodies are quite different, and they are two TSHR inhibitory antibodies with completely different sequences. The TSHR inhibitory monoclonal antibody screened in the present application with the closest evolutionary distance to K1-70 is CHK11, and its inhibitory activity is 75% at an antibody concentration of 2 μg / mL, indicating that the inhibitory efficiency of K1-70 on TSHR is lower than that of CHK36, the antibody with the strongest inhibitory activity screened in this screening.

[0147] Orbital fibroblast (OF cell) inhibition effect evaluation: OF cells were cultured in a 10 cm dish containing 10% FBS in DMEM, and when they reached 70%, they were subcultured into a 12-well plate and cultured in DMEM containing 1% FBS for 16 hours. After adding 50 ng / ml of inhibitory antibody SH1, continue to culture for 24 hours. Collect cells to extract RNA and detect the expression level of fibrosis-related gene mRNA by RT-qPCR. The results show that TSHR inhibitory monoclonal antibody CHK36 can significantly inhibit the expression of fibrosis marker collagen 1A2 (COL1A2) mRNA (Figure 4).

[0148] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0149] As used herein, the terms "comprises", "comprising", "includes", "including" or "contains", "containing" are open-ended, i.e., including one or more of the indicated steps or elements but not excluding others.

[0150] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0151] The protection scope of the present application is not limited to the above-mentioned embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application and are protected by the appended claims.

Claims

1. A panel of fully human TSH receptor blocking monoclonal antibodies or antigen binding fragments thereof, characterized in that, The full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof binds to the TSH receptor and blocks the binding of TSH to the TSH receptor; the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, and the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are respectively shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28; The light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, the amino acid sequence of L-CDR1 is selected from one of SEQ ID NO: 29-34; the amino acid sequence of L-CDR2 is selected from one of SEQ ID NO: 35-38; and the amino acid sequence of L-CDR3 is selected from one of SEQ ID NO: 39-44.

2. The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO: 1, 3, 5, 7, 9, 11 or has at least 85% sequence identity with one of SEQ ID NO: 1, 3, 5, 7, 9, 11; The amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or has at least 85% sequence identity with one of SEQ ID NO: 2, 4, 6, 8, 10, 12.

3. The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, characterized in that, The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), Fav, dsFv, sc(Fv)2, single domain antibody (dAb).

4. The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, characterized in that, The full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof is an antagonist of TSH; and / or is an antagonist of thyroid stimulating antibody.

5. The substance as claimed in any of the preceding claims, characterized in that The substance comprises: (1) a preparation, a drug or a pharmaceutical composition comprising the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2; (2) a reagent or kit containing the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2; (3) a nucleotide encoding the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, and the nucleotide sequence thereof comprises: (a) the nucleotide sequence encoding the heavy chain variable region is shown in one of SEQ ID NO: 13, 15, 17, 19, 21, 23; and the nucleotide sequence encoding the light chain variable region is shown in one of SEQ ID NO: 14, 16, 18, 20, 22, 24; (b) a nucleotide sequence having at least 85% sequence identity with one of SEQ ID NO: 13-SEQ ID NO:

24. (c) a nucleotide sequence that is the addition, substitution, deletion or insertion of one or several nucleotides to the nucleotide sequence as shown in SEQ ID NO: 13-SEQ ID NO: 24; (d) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the preceding (a), (b) or (c) or the full-length complement thereof; or, (e) a nucleotide sequence that differs from the nucleotide sequence of the preceding (a), (b), (c), (d) due to the degeneracy of the genetic code; wherein the nucleotide sequence of the nucleic acid or a portion thereof encodes an antibody V H domain and an antibody V L domain or is selected from the CDRs set forth as SEQ ID NO: 26-SEQ ID NO: 44; (4) a vector comprising the nucleotide of (3); (5) a host cell comprising the nucleotide of (3) and / or the vector of (4); (6) a cell that is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, and / or the nucleotide of (3), and / or the vector of (4); and / or, an isolated cell that expresses the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2; and / or, an isolated cell that secretes the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2; (7) a composition comprising a determined concentration of TSH receptor autoantibodies and comprising a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; (8) a pharmaceutical composition for administration to a mammalian subject for the treatment of a thyroid-related condition, said pharmaceutical composition comprising a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 and a pharmaceutically acceptable carrier; wherein said thyroid-related condition is selected from the group consisting of hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema.

6. The substance of claim 5, wherein, The pharmaceutical composition is suitable for administration to a human.

7. The substance of claim 5, wherein, The pharmaceutical composition comprises one or more additional thyroid-stimulating hormone receptor antagonists.

8. The substance of claim 5, wherein, The pharmaceutical composition comprises a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 and any pharmaceutically acceptable carrier, adjuvant or vehicle.

9. The substance of claim 5, wherein, The pharmaceutical composition is administered by oral administration, parenteral administration, administration by spray inhalation, topical administration, administration by eye drops or eye ointment, rectal administration, nasal administration, buccal administration, vaginal administration, administration via an implanted reservoir.

10. The substance of claim 5, wherein, The pharmaceutical composition is formulated in the form of a capsule, tablet, aqueous suspension, solution, rectal suppository, enema, ointment, lotion, cream, nasal spray, inhalant.

11. The substance of claim 5, wherein, The pharmaceutical composition is used to treat a thyroid-related condition in an injectable form.

12. The substance of claim 5, wherein, The pharmaceutical composition is used to treat pretibial myxedema in a topical administration form; and / or, The pharmaceutical composition is used to treat a thyroid-related condition in an injectable form. The pharmaceutical composition is used for treating Graves' ophthalmopathy in the form of intravenous injection preparation or eye drops.

13. Any of the following methods, characterized in that, The method comprises: (1) A preparation method of a fully human TSH receptor blocking monoclonal antibody or an antigen binding fragment thereof, and the preparation method specifically comprises the following steps: Step 1: Sorting TSHR-specific plasma cells and memory B cells from peripheral blood of patients with high TBAb activity, performing single cell RNA extraction and cDNA synthesis, and verifying the sorted single cells by nested PCR amplification of heavy chain H, light chain lambda and light chain kappa, and selecting single cells with positive heavy chain and light chain for subsequent cloning; Step 2: In vitro amplification by nested PCR to clone the BCR heavy chain and light chain of all single B cells into an expression heavy chain vector AbVec-IGHG1, a lambda light chain expression vector AbVec-hIgKappa or a lambda light chain expression vector AbVec-hIgLambda; Step 3: After successfully obtaining the heavy chain and light chain recombinant plasmid, sequencing and alignment analysis are performed on the obtained candidate clones to determine the number of nucleotide and amino acid sequences of the obtained candidate antibodies; the heavy chain and light chain expression plasmid is transfected to express the monoclonal antibody in vitro; the antigen binding capacity and antibody blocking activity are further verified, and finally the antibody combination specific to the target antigen, i.e., the fully human TSH receptor blocking monoclonal antibody, is obtained; (2) A method for treating a thyroid-related disorder in a mammalian subject or in a cell derived from the subject, the method comprising contacting the subject or the cell with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2; (3) A method for inhibiting thyroid stimulating antibodies from stimulating TSH receptors in a thyroid of a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2; (4) A method for inhibiting thyroid stimulating autoantibodies from binding to TSH receptors outside the thyroid in a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2; (5) A method for treating thyroid cancer or metastatic thyroid cancer in a thyroid of a subject or in a thyroid cell derived from the subject, the method comprising contacting the cancer cell with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2, with the purpose of inhibiting constitutive thyroid stimulating hormone receptor activity in the cell; (6) A method for treating hyperthyroidism caused by constitutive thyroid activity in a subject or in a thyroid cell derived from the subject, characterized in that the method comprises contacting the subject or the cell with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2, with the purpose of inhibiting such hyperthyroidism. (7) A method for identifying a molecule that can inhibit the binding of thyroid stimulating antibodies to TSH receptor, said method comprising providing as a reference at least one fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2; (8) A method for identifying a molecule that can inhibit the binding of thyroid blocking antibodies to TSH receptor, said method comprising providing as a reference at least one fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2.

14. The method of claim 13, wherein, The thyroid related disorder is selected from the group consisting of hyperthyroidism, thyroid associated ophthalmopathy (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema.

15. The method of claim 13, wherein, The subject is a human.

16. The method of claim 13, wherein, In method (3), the method prevents the binding of thyroid stimulating antibodies to TSHR.

17. The method of claim 13, wherein, In method (4), wherein the ectopic TSH receptor is located in retro-orbital tissue and / or pretibial tissue of the subject; and / or, The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof blocks the binding of TSHR autoantibodies to ectopic TSHR.

18. The method of claim 13, wherein, In method (5), the method prevents or delays the regrowth of thyroid cancer cells.

19. The method of claim 13, wherein, In method (7), the method selects a molecule to be tested that can prevent the binding of thyroid stimulating antibodies to TSHR.

20. The method of claim 13, wherein, In method (8), the method selects a molecule that can prevent the binding of thyroid blocking antibodies to TSHR.

21. Use of any one of the preceding claims, characterized in that The uses include: (1) Use of a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2 for treating a thyroid related disorder; (2) Use of a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2 for the manufacture of a medicament for treating a thyroid related disorder; (3) Use of a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2 for the manufacture of a reagent or kit for detecting TSH receptor antibodies; (4) Use of a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2 for the manufacture of a reagent or kit or product for detecting hyperthyroidism, thyroid associated ophthalmopathy; (5) Use of a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, or a substance according to claim 5, or a method according to claim 13 for the manufacture of a preparation, medicament or pharmaceutical composition for detecting TSH receptor antibodies, for the manufacture of a medicament for treating hyperthyroidism, thyroid associated ophthalmopathy, for the manufacture of a medicament for inhibiting thyroid hyperplasia and / or thyroid hormone production, for the manufacture of a TSH receptor blocking medicament, for the manufacture of a medicament for antagonizing the activating effect of TSH on TSH receptor.

22. The use according to claim 21, characterized in that, The thyroid-related disorder is selected from the group consisting of hyperthyroidism, thyroid-associated ophthalmopathy (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema.

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