SH1 homologous cluster fully human TSH receptor blocking monoclonal antibody group as well as preparation method and application of SH1 homologous cluster fully human TSH receptor blocking monoclonal antibody group

Abstract

The invention discloses a group of blocking monoclonal antibodies of an SH1 homologous cluster fully human thyrotropin receptor (TSHR) (a TSH receptor, TSHR) as well as a preparation method and application of the blocking monoclonal antibodies of the SH1 homologous cluster fully human thyrotropin receptor (TSHR). 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 receptor 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. 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, and can effectively block signal transduction after the TSH is combined with the receptor; the synthesis and secretion of thyroid hormone are inhibited; expression and fibrosis of effector cell orbital fibroblasts TSHR for inhibiting thyroid-related eye diseases are remarkably relieved, and wide application prospects are achieved in treatment of Graves' diseases and diseases such as thyroid eye diseases caused by hyperthyroidism.

Description

Technical Field

[0001] The present invention belongs to the fields of monoclonal antibody technology and human antibody drugs, and relates to a group of fully human TSH receptor-blocking monoclonal antibodies, a preparation method thereof and applications thereof. Background Art

[0002] The thyroid-stimulating hormone receptor (TSHR) belongs to the seven-transmembrane G protein-coupled receptor and mainly exists on the cell membrane of thyroid follicular epithelial cells. TSHR has a large extracellular domain, which is composed of a leucine-rich domain and a "hinge" region. Abnormal functions of TSHR may lead to the occurrence of thyroid diseases. After thyroid-stimulating hormone (TSH) binds to and activates TSHR, downstream signal transduction can be mediated by the Gs and Gq / 11 pathways, regulating iodine transport and absorption, iodine organification, etc., and participating 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 TSHR can be divided into an α-subunit and a β-subunit, and the α-subunit is prone to shedding. Against the background of genetic susceptibility, under the action of external environmental factors such as infection and mental trauma, the TSHR peptide segment can serve as an exposed autoantigen, inducing the production of autoantibodies in the body, namely thyrotropin receptor antibody (TRAb), and participating in the occurrence and development of autoimmune thyroid disease (AITD), mainly including Graves' disease (GD) and Hashimoto's thyroiditis.

[0003] Graves' disease is an organ-specific autoimmune disease caused by the combined action of genetics and environment, with increased secretion of thyroid hormones, and is the most common cause of hyperthyroidism. There are a large number of GD patients, and the incidence in the population is about 0.2-2%. Like other autoimmune diseases, GD is prone to occur in women of childbearing age, and the female incidence is about 5-10 times that of men. In recent years, with the change of living environment, the incidence of hyperthyroidism has been on the rise. If the symptoms of hyperthyroidism are not controlled in time, it can affect the heart, leading to arrhythmia or heart failure, etc. In women of childbearing age, it can cause menstrual disorders, difficulty in getting pregnant, miscarriage, etc. In addition, hyperthyroidism can mostly cause mental abnormalities such as nervousness, anxiety, irritability, etc., affecting the learning and life of patients, and in severe cases, it can even cause mental disorders. At present, the treatment of GD mainly includes drugs, radioactive iodine and surgical treatment. The former has a long course of treatment, is difficult for patients to adhere to, and has a high recurrence rate. About 60-70% of patients will relapse. Clinically, the treatment of GD 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 a history of nearly 70 years. Except in the United States, doctors around the world regard ATD as the first choice for the treatment of Graves' disease. However, after regular and systematic treatment, only some patients with hyperthyroidism can be cured, and the side effects are relatively large. A considerable number of patients will relapse after a certain period of time. When relapsed, the condition worsens, and generally radioactive iodine or surgery is needed for treatment, which greatly increases the economic and psychological burden on patients. In addition, radioactive iodine 131 nuclide treatment is prone to cause 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 clinically.

[0004] TRAb is the general term for antibodies produced by the body against TSHR. It is a group of polyclonal antibodies and can be divided into TSH receptor-stimulating antibody (TSH-stimulating antibody, TSAb), TSH receptor-blocking antibody (TSH-stimulating blocking antibody, TBAb), and neutral antibody. Their recognition epitopes on TSHR are not the same. The recognition epitopes of TSAb, TBAb, and neutral antibody are concentrated in the amino-terminal (N-terminal), carboxyl-terminal (C-terminal), and hinge region of the extracellular domain of TSHR, respectively. Among them, the thyroid-stimulating antibody TSAb binds to TSHR on the thyroid follicular epithelial cell membrane, produces a biological effect similar to TSH, causes hyperthyroidism, and is the direct pathogenic cause of Graves’ disease. Through the stimulating G protein-coupled effect, TSAb further activates adenylate cyclase (AC) to stimulate the production of cAMP. The AC-cAMP pathway is in a continuously active state, stimulating the proliferation of thyroid cells. The thyroid synthesizes and secretes excessive thyroid hormones, namely triiodothyronine (T3) and thyroxine (T4). TSH is competitively inhibited and cannot normally exert its feedback regulatory effect on T3 and T4, resulting in continuous increases in T3 and T4, causing a series of reactions in the body, and further leading to hyperfunction of thyroid cells. At the histological level, the thyroid of GD patients shows thickened and hypertrophied follicular cells. The gland shows typical lymphocyte infiltration of T cells and B cells, has the characteristics of thyroiditis, and occasionally has a little apoptosis and a certain degree of follicular destruction. While TBAb binds to TSH to block the binding of TSH to the receptor, inhibiting thyroid hyperplasia and thyroid hormone production. Both stimulating and blocking antibodies coexist in the body of GD patients, and the final result of their thyroid function depends on which antibody predominates. This makes it a new and effective treatment plan for GD to externally supplement a certain dose of blocking antibody to bind to TSHR and thus improve the pathophysiological effects of autoantibodies.

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

[0006] Thyroid eye disease is a common orbital disease in adults and an autoimmune disease closely related to Graves' disease. It can manifest as congestion and edema of the eyelids and conjunctiva, fibrosis and fatification of orbital tissues, resulting in impaired eye movement, strabismus and diplopia, which can cause blindness and disability. The diagnosis and treatment are difficult, seriously affecting the quality of life of patients. China is one of the countries with a high incidence of GD in the world. The prevalence of TED in GD patients of Asian ethnicity is as high as 45%. The TED patient group is huge, and their visual health and quality of life have been seriously affected. As a problem that has plagued the global medical community for two centuries, there is currently a lack of effective treatment methods for TED. At present, although there are various treatment options for TED, many patients do not respond well to existing drugs or cannot tolerate the side effects. High-dose glucocorticoid pulse therapy, although it is obvious in reducing inflammation, has no obvious effect on proptosis and diplopia, and has great side effects when used for a long time, such as hypertension, diabetes, osteoporosis, gastric ulcer, etc. Some patients are insensitive to glucocorticoids, and the effective rate is only 50-75%. In addition, there are also disadvantages such as obvious short-term effects but easy recurrence after drug withdrawal. Immunosuppressants can reduce ocular inflammation by inhibiting the overreaction of the immune system, but they will cause serious side effects, such as an increased risk of infection and damage to liver and kidney functions. Therefore, the market urgently needs new, safe and effective drugs.

[0007] Orbital fibroblasts (OF) are effector cells of the autoimmune response in TED. There are thyroid-stimulating hormone receptors (TSHR) and insulin-like growth factor-1 receptors (IGF-1R) on their surfaces. Ectopic overexpression of TSHR is found in CD34+ fibroblasts in the retrobulbar tissue of TED patients. The humoral immune response in TED begins with the abnormal recognition of the autoantigen thyrotropin receptor (TSHR), and then the activated B cells produce TRAb. IGF-1R is another possible TED autoantigen, but it is not clear whether there are autoantibodies that directly activate IGF-1R. Some studies have shown that the signal transduction pathways mediated by IGF-1R and TSHR bind to each other, resulting in a large amount of secretion of hyaluronic acid (HAS). Currently, the antibody drug (Tepezza) targeting IGF-1R was approved by the FDA for the treatment of TED in 2020. After treatment, it can significantly improve proptosis and diplopia. In nearly 40% of patients with proptosis, the degree of proptosis can basically return to normal. Although the IGF-1R antibody drug has significant curative effects, due to the widespread expression of IGF-1R in human organs, many significant side effects have been reported after large-scale clinical application, especially irreversible deafness and the impact on the reproductive system. Since both stimulating and blocking antibodies coexist in GD patients, the final result of their thyroid function depends on which antibody predominates. This makes the exogenous supplementation of a certain dose of blocking antibody to inhibit TSHR a new and effective treatment plan for thyroid eye disease.

[0008] Humanized monoclonal antibodies can be divided into murine-humanized monoclonal antibodies and fully human monoclonal antibodies. Although murine-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 also be reduced to a certain extent during the remodeling process. In contrast, fully human monoclonal antibodies directly amplify antibody genes from human single B cells, obtain a large number of naturally paired antibody heavy and light chain genes by amplifying the antibody genes of isolated single plasma cells or memory B cells, and then screen for antibodies with antigen specificity and neutralizing activity by expressing the paired antibody heavy and light chain genes. This method has the advantages of being fast, high-throughput, and requiring a small amount of cells. The fully human antibodies prepared by this method retain rich gene diversity and natural pairing of the heavy and light chain variable regions, and have great advantages. Currently, the preparation of fully human antibodies against influenza, anthrax virus, and pneumococcus is based on this technology. Summary of the Invention

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

[0010] In the present invention, the term "TSHR" refers to the full-length human thyroid-stimulating hormone receptor having the amino acid sequence shown in SEQ ID NO: 29, or a variant or fragment highly homologous to the thyroid-stimulating hormone receptor. Preferably, such a variant and fragment have 70-99.9% homology with the amino acid sequence shown in SEQ ID NO: 29.

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

[0012] Among them, the heavy chain variable region contains H-CDR1, H-CDR2, and H-CDR3, and the amino acid sequence of H-CDR1 is selected from one of SEQ ID NO: 30-33; the amino acid sequence of H-CDR2 is selected from one of SEQ ID NO: 34-36; the amino acid sequence of H-CDR3 is selected from one of SEQ ID NO: 37-40;

[0013] Among them, the light chain variable region contains L-CDR1, L-CDR2 and L-CDR3. The amino acid sequence of L-CDR1 is selected from one of SEQ ID NO: 41 to 42; the amino acid sequence of L-CDR2 is selected from one of SEQ ID NO: 43 to 45; the amino acid sequence of L-CDR3 is selected from one of SEQ ID NO: 46 to 48.

[0014] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 37; or,

[0015] the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 38; or,

[0016] the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 39; or,

[0017] the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 40;

[0018] Preferably, the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region are respectively as shown in SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 46; or,

[0019] the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region are respectively as shown in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 47; or,

[0020] the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region are respectively as shown in SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 48.

[0021] More preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:37 respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:46 respectively.

[0022] More preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:37 respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:47 respectively.

[0023] More preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO:31, SEQ ID NO:35 and SEQ ID NO:38 respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:46 respectively.

[0024] More preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO:32, SEQ ID NO:35 and SEQ ID NO:39 respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:46 respectively.

[0025] More preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO:32, SEQ ID NO:35 and SEQ ID NO:39 respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO:41, SEQ ID NO:45 and SEQ ID NO:48 respectively.

[0026] Further preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO:33, SEQ ID NO:36, and SEQ ID NO:40 respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:46 respectively.

[0027] Among them, the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO:1, 3, 5, 7, 9, 11, 13 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% or more sequence identity with one of SEQ ID NO:1, 3, 5, 7, 9, 11, 13;

[0028] Among them, the amino acid sequence of the light chain variable region is selected from one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14 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% or more sequence identity with one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14.

[0029] 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).

[0030] In the present invention, 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 its antigen-binding fragment are shown as SEQ ID NO:15 - SEQ ID NO:28.

[0031] Preferably, 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 its antigen-binding fragment have 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:15 - SEQ ID NO:28.

[0032] SEQ ID NO: 1 (Amino acid sequence of SH1_1)

[0033] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln ProGly Arg Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Lys Tyr AlaMet His Trp Ala Arg Gln Ala Pro GlyLys Gly Leu Gln Trp Val Ala Ala Ile SerTyr Asp Gly Ser His Thr Tyr Tyr Ala Asp Cys Val Lys SerArg Phe Thr Ile SerArg Asp Asn Ser Lys Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Arg Asp GluAspThr Ala Val Tyr Phe Cys Gly Lys Gly Ser Tyr Asp Thr Ser Gly Tyr Phe Leu AsnTrp Phe AspPro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[0034] (VHSEVQLVESGGGVVQPGRSLRLSCAASGFTFRKYAMHWARQAPGKGLQWVAAIS YDGSHTYYADCVKSRFTISRDNSKNTLSLQMNSLRDEDTAVYFCGKGSYDTSGYFLNWFDP WGQGTLVTVSS)

[0035] SEQ ID NO: 2 (Amino acid sequence of SH1_2)

[0036] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr ThrTyr Val Ser Trp Tyr Gln Gln His Pro Gly GluAlaPro Glu Leu Met Ile Tyr Asp ValSer Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser LysSer Gly Asn ThrAla Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr CysCysSer Tyr Ala Gly Ser Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr ValLeu Gly Gln Pro LysAlaAla Pro Ser Ala Thr Leu Phe His

[0037] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVFGGGTKLTVLGQPKA APSATLFH)

[0038] SEQ ID NO: 15 (Nucleotide sequence of SH1_3)

[0039] GTACATTCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCTGCCTCTGGATTCACCTTCAGAAAGTATGCCATGCACTGGGCCCGCCAGGCCCCAGGCAAGGGGCTGCAGTGGGTGGCAGCTATATCATATGATGGAAGTCACACATACTACGCAGACTGCGTGAAGAGTCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTCTCTGCAAATGAACAGCCTGAGAGATGAGGACACGGCTGTGTATTTCTGTGGGAAAGGATCCTATGATACAAGCGGTTATTTCTTGAATTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0040] SEQ ID NO: 16 (Nucleotide sequence of SH1_4)

[0041] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCAC

[0042] SEQ ID NO: 3 (Amino acid sequence of SH2_1)

[0043] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Lys Tyr Ala Met His Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val Ala Ala Ile Ser Tyr Asp Gly Ser His Thr Tyr Tyr Ala Asp Cys Val Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Phe Cys Gly Lys Gly Ser Tyr Asp Thr Ser Gly Tyr Phe Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[0044] (VHSEVQLVESGGGVVQPGRSLRLSCAASGFTFRKYAMHWARQAPGKGLQWVAAIS YDGSHTYYADCVKSRFTISRDNSKNTLSLQMNSLRDEDTAVYFCGKGSYDTSGYFLNWFDP WGQGTLVTVSS)

[0045] SEQ ID NO: 4 (Amino acid sequence of SH2_2)

[0046] Ser Leu Ser Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser ProGly Gln Thr Ala Arg IleThr Cys Ser Gly Gly Arg Leu Gly Asp Thr Phe Ala SerTrp Tyr Gln Gln Arg Pro Gly Gln Ala ProVal Leu Val Ile Tyr Arg Asp Thr MetArg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser GlyHis Ala Ala ThrLeu Thr Ile Ser Gly Thr Gln Thr Met Asp Glu Ala Asp Tyr Tyr Cys Gln ValTrpAsp Thr Ser Ala Ser Ala His Val Phe Gly Thr Gly Thr Thr Val Thr Val LeuGly Gln Pro Lys AlaAsn Pro Thr Val Thr Leu Phe Pro

[0047] (SLSQPVLTQPPSVSVSPGQTARITCSGGRLGDTFASWYQQRPGQAPVLVIYRDTMRPSGIPERFSGSNSGHAATLTISGTQTMDEADYYCQVWDTSASAHVFGTGTTVTVLGQPKANPTVTLFP)

[0048] SEQ ID NO: 17 (Nucleotide sequence of SH2_3)

[0049] GTACATTCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCTGCCTCTGGATTCACCTTCAGAAAGTATGCCATGCACTGGGCCCGCCAGGCCCCAGGCAAGGGGCTGCAGTGGGTGGCAGCTATATCATATGATGGAAGTCACACATACTACGCAGACTGCGTGAAGAGTCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTCTCTGCAAATGAACAGCCTGAGAGATGAGGACACGGCTGTGTATTTCTGTGGGAAAGGATCCTATGATACAAGCGGTTATTTCTTGAATTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0050] SEQ ID NO: 18 (Nucleotide sequence of SH2_4)

[0051] TCTCTCTCGCAGCCTGTGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGAATCACCTGCTCTGGGGGAAGATTGGGGGATACATTTGCTTCCTGGTATCAACAGAGGCCAGGCCAGGCCCCTGTGTTGGTCATCTATCGTGATACGATGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGCTCCAACTCTGGGCACGCAGCCACTCTGACCATCAGCGGGACGCAGACTATGGATGAGGCTGACTACTACTGTCAGGTGTGGGACACCAGCGCCAGCGCACATGTCTTCGGAACTGGGACCACGGTCACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTCCCG

[0052] SEQ ID NO: 5 (Amino acid sequence of SH3_1)

[0053] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Lys Tyr Ala Met His Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val Ala Ala Ile Ser Tyr Asp Gly Ser His Thr Tyr Tyr Ala Asp Cys Val Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Phe Cys Gly Lys Gly Ser Tyr Asp Thr Ser Gly Tyr Phe Leu Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[0054] (VHSEVQLVESGGGVVQPGRSLRLSCAASGFTFRKYAMHWARQAPGKGLQWVAAISYDGSHTYYADCVKSRFTISRDNSKNTLSLQMNSLRDEDTAVYFCGKGSYDTSGYFLNWFDPWGQGTLVTVSS)

[0055] SEQ ID NO: 6 (Amino acid sequence of SH3_2)

[0056] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr ThrTyr Val Ser Trp Tyr Gln Gln His Pro Gly Glu Ala Pro Glu Leu Met Ile Tyr AspVal Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly AsnThr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr CysCys Ser Tyr Ala Gly Ser Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr ValLeu Gly Gln Pro Lys Ala Ala Pro Ser Ala Thr Leu Phe Pro

[0057] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVFGGGTKLTVLGQPKAAPSATLFP)

[0058] SEQ ID NO: 19 (SH3_3 nucleotide sequence)

[0059] GTACATTCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCTGCCTCTGGATTCACCTTCAGAAAGTATGCCATGCACTGGGCCCGCCAGGCCCCAGGCAAGGGGCTGCAGTGGGTGGCAGCTATATCATATGATGGAAGTCACACATACTACGCAGACTGCGTGAAGAGTCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTCTCTGCAAATGAACAGCCTGAGAGATGAGGACACGGCTGTGTATTTCTGTGGGAAAGGATCCTATGATACAAGCGGTTATTTCTTGAATTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0060] SEQ ID NO: 20 (Nucleotide sequence of SH3_4)

[0061] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCA

[0062] SEQ ID NO: 7 (Amino acid sequence of SH7_1)

[0063] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Leu Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val Ser Arg Ile Asn Thr Asp Gly Ser Arg Ile Asp Tyr Ala Gly Ser Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Thr Val Tyr Tyr Cys Val Asn Gln Leu Leu Gly Thr Thr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[0064] (VHSEVQLVESGGGLVQPGGSLRLSCTASGFTLSTYGMHWVRQAPGKGLVWVSRINTDGSRIDYAGSVKGRFTASRDNAKNTLFLQMNSLRAEDTTVYYCVNQLLGTTNYWGQGTLVTVSS)

[0065] SEQ ID NO: 8 (Amino acid sequence of SH7_2)

[0066] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr ThrTyr Val Ser Trp Tyr Gln Gln His Pro Gly GluAlaPro Glu Leu Met Ile Tyr Asp ValSer Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser LysSer Gly Asn ThrAla Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr CysCysSer Tyr Ala Gly Ser Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr ValLeu Gly Gln Pro LysAlaAla Pro Ser Ala Thr Leu Phe His

[0067] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVFGGGTKLTVLGQPKAAPSATLFH)

[0068] SEQ ID NO: 21 (Nucleotide sequence of SH7_3)

[0069] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCCTCAGTACCTACGGGATGCACTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTATCAACACTGATGGGAGTAGGATAGACTACGCGGGCTCCGTGAAGGGCCGATTCACCGCCTCCAGAGACAACGCCAAGAACACACTGTTTCTGCAAATGAACAGTCTGAGAGCCGAGGACACGACTGTCTATTATTGCGTAAACCAATTACTGGGAACTACTAACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0070] SEQ ID NO: 22 (Nucleotide sequence of SH7_4)

[0071] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCAC

[0072] SEQ ID NO: 9 (Amino acid sequence of SH18_1)

[0073] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Ser Ser Ala Ala Thr Val Phe Tyr Ala Asp Ser Val Gln Gly Arg Leu Thr Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gln Phe Thr Gly Asn Ile His Tyr Phe Asp His Trp Gly Gln Gly Asp Leu Val Thr Val Ser Ser

[0074] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISS SAATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCARDQFTGNIHYFDHWGQ GDLVTVSS)

[0075] SEQ ID NO: 10 (Amino acid sequence of SH18_2)

[0076] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr ThrTyr Val Ser Trp Tyr Gln Gln His Pro Gly GluAlaPro Glu Leu Met Ile Tyr Asp ValSer Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser LysSer Gly Asn ThrAla Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr CysCysSer Tyr Ala Gly Ser Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Ser ValLeu Arg Gln Pro LysAlaAla Pro Ser Ala Thr Leu Phe Pro

[0077] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVFGGGTKLSVLRQPKA APSATLFP)

[0078] SEQ ID NO: 23 (Nucleotide sequence of SH18_3)

[0079] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGCGAGAGATCAATTTACCGGCAACATCCACTACTTTGACCACTGGGGCCAGGGAGACCTGGTCACCGTCTCCTCA

[0080] SEQ ID NO: 24 (Nucleotide sequence of SH18_4)

[0081] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGTCCGTCCTACGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCA

[0082] SEQ ID NO: 11 (Amino acid sequence of SH19_1)

[0083] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Ser Ser Ala Ala Thr Val Phe Tyr Ala Asp Ser Val Gln Gly Arg Leu Thr Ile Ser Arg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gln Phe Thr Gly Asn Ile His Tyr Phe Asp His Trp Gly Gln Gly Asp Leu Val Thr Val Ser Ser

[0084] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISS SAATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCARDQFTGNIHYFDHWGQ GDLVTVSS)

[0085] SEQ ID NO: 12 (Amino acid sequence of SH19_2)

[0086] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr ThrTyr Val Ser Trp Tyr Gln Gln His Pro Gly GluAla Pro Glu Leu Met Ile Tyr AspVal Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly SerLys Ser Gly AsnThr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr TyrCysCys Ser Tyr Ala Gly Ser Asp Thr Trp Val Phe Gly Gly Gly Thr Lys Leu ThrVal Leu Gly Gln ProLys Ala Ala Pro Ser Ala Thr Leu Phe Pro

[0087] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS NRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSDTWVFGGGTKLTVLGQPKA APSATLFP)

[0088] SEQ ID NO: 25 (SH19_3 nucleotide sequence)

[0089] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGCGAGAGATCAATTTACCGGCAACATCCACTACTTTGACCACTGGGGCCAGGGAGACCTGGTCACCGTCTCCTCA

[0090] SEQ ID NO: 26 (Nucleotide sequence of SH19_4)

[0091] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCGACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCA

[0092] SEQ ID NO: 13 (Amino acid sequence of SH63_1)

[0093] Val His Ser Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Ile Tyr Cys Ser Ser Thr Ser Cys Tyr Arg Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[0094] (VHSEVQLVESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWIS AYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDRIYCSSTSCYRWF DPWGQGTLVTVSS)

[0095] SEQ ID NO: 14 (Amino acid sequence of SH63_2)

[0096] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr ThrTyr Val Ser Trp Tyr Gln Gln His Pro Gly GluAlaPro Glu Leu Met Ile Tyr Asp ValSer Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser LysSer Gly Asn ThrAla Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr CysCysSer Tyr Ala Gly Ser Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr ValLeu Gly Gln Pro LysAlaAla Pro Ser Ala Thr Leu Phe Pro

[0097] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVFGGGTKLTVLGQPKA APSATLFP)

[0098] SEQ ID NO: 27 (Nucleotide sequence of SH63_3)

[0099] GTACATTCTGAGGTGCAGCTGGTGGAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCGGATATATTGTAGTAGTACCAGCTGCTATAGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0100] SEQ ID NO: 28 (Nucleotide sequence of SH63_4)

[0101] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCA

[0102] SEQ ID NO: 29 (Amino acid sequence of human TSHR)

[0103] MRPADLLQLVLLLDLPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMGYKFLRIVVWFVSLLALLGNVFVLLILLTSHYKLNVPRFLMCNLAFADFCMGMYLLLIASVDLYTHSEYYNHAIDWQTGPGCNTAGFFTVFASELSVYTLTVITLERWYAITFAMRLDRKIRLRHACAIMVGGWVCCFLLALLPLVGISSYAKVSICLPMDTETPLALAYIVFVLTLNIVAFVIVCCCYVKIYITVRNPQYNPGDKDTKIAKRMAVLIFTDFICMAPISFYALSAILNKPLITVSNSKILLVLFYPLNSCANPFLYAIFTKAFQRDVFILLSKFGICKRQAQAYRGQRVPPKNSTDIQVQKVTHEMRQGLHNMEDVYELIENSHLTPKKQGQISEEYMQTVL

[0104] Table 1: CDR regions of the heavy chain (H) and light chain (L) of the fully human TSH receptor-blocking monoclonal antibody of the SH1 homologous cluster

[0105]

[0106]

[0107] The present invention includes a group of fully human TSH receptor-blocking monoclonal antibodies or their antigen-binding fragments, namely SH1, SH3, SH2, SH7, SH18, SH19, SH63. This group of antibodies has a high homology with SH1, so it is named a group of SH1 homologous cluster fully human TSH receptor-blocking monoclonal antibodies or their antigen-binding fragments. In the above table, K1-70 is a reported TSH receptor-blocking monoclonal antibody with blocking activity. In comparison, the sequences provided by the present invention are very different and have no homology at all.

[0108] Preferably, the present invention provides a group of fully human TSH receptor-blocking monoclonal antibodies or their antigen-binding fragments, named SH1 homologous cluster fully human TSH receptor-blocking monoclonal antibodies or their antigen-binding fragments.

[0109] In the present invention, the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment is an antagonist of TSH.

[0110] In the present invention, the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment is an antagonist of thyroid-stimulating antibody.

[0111] In the present invention, the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment respectively contains a V H region (heavy chain variable region), and the V H region contains complementarity-determining regions (CDRs) with amino acid sequences as shown in SEQ ID NO: 30, 34, 37 (see Table 1) or complementarity-determining regions CDRs with amino acid sequences as shown in SEQ ID NO: 31, 35, 38 (see Table 1) or complementarity-determining regions CDRs with amino acid sequences as shown in SEQ ID NO: 32, 35, 39 (see Table 1) or complementarity-determining regions CDRs with amino acid sequences as shown in SEQ ID NO: 33, 36, 40 (see Table 1).

[0112] In the present invention, the antibody respectively contains a V L region (light chain variable region), and the V L region contains CDRs with amino acid sequences as shown in SEQ ID NO: 41, 43, 46 (see Table 1) or complementarity-determining regions CDRs with amino acid sequences as shown in SEQ ID NO: 42, 44, 47 (see Table 1) or complementarity-determining regions CDRs with amino acid sequences as shown in SEQ ID NO: 41, 45, 48 (see Table 1).

[0113] In the present 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.

[0114] The fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment 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, reducing hyperthyroidism caused by various reasons; can significantly reduce goiter caused by hyperthyroidism and other reasons after binding to the TSH receptor; and alleviates the inflammation, edema, hyperplasia, etc. of thyroid-associated ophthalmopathy.

[0115] The thyroid-associated ophthalmopathy (TAO) described in the present invention is also known as thyroid eye disease (TED).

[0116] The present invention also provides a preparation, drug or pharmaceutical composition, which contains the above-mentioned fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment.

[0117] The present invention also provides a reagent or kit, which contains the above-mentioned fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment.

[0118] The present invention also provides a nucleotide encoding the above-mentioned fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment, and its nucleotide sequence is one of the following sequences:

[0119] (a) The nucleotide sequence encoding the heavy chain variable region is shown as one of SEQ ID NO: 15, 17, 19, 21, 23, 25, 27; the nucleotide sequence encoding the light chain variable region is shown as one of SEQ ID NO: 16, 18, 20, 22, 24, 26, 28;

[0120] (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 with SEQ ID NO: 15 - SEQ ID NO: 28;

[0121] (c) A nucleotide sequence obtained by adding, substituting, deleting or inserting one or several nucleotides to the nucleotide sequence shown in SEQ ID NO: 15 - SEQ ID NO: 28;

[0122] (d) A nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the foregoing (a), (b) or (c) or its full-length complement; or,

[0123] (e) A nucleotide sequence that is different from the nucleotide sequences in (a), (b), (c), and (d) above due to the degeneracy of the genetic codon;

[0124] Wherein, the nucleotide sequence of the nucleotide or a part thereof encodes an antibody V H domain; and antibody V L domain or a CDR selected from those shown in SEQ ID NO: 30 - SEQ ID NO: 48 (see Table 1).

[0125] The present invention also provides a vector, which contains the nucleotide as described above.

[0126] The present invention also provides a host cell, which contains the nucleotide as described above, and / or the vector as described above.

[0127] The present invention also provides a cell, which is an isolated cell containing the fully human TSH receptor - blocking monoclonal antibody or its antigen - binding fragment as described above, and / or the nucleotide as described above, and / or the vector as described above.

[0128] The present invention also provides a cell, which is an isolated cell expressing the fully human TSH receptor - blocking monoclonal antibody or its antigen - binding fragment as described above.

[0129] The present invention also provides a cell, which is an isolated cell secreting the fully human TSH receptor - blocking monoclonal antibody or its antigen - binding fragment as described above.

[0130] The present invention also provides a composition, which contains a TSH receptor autoantibody at a determined concentration and contains a fully human TSH receptor - blocking monoclonal antibody or its antigen - binding fragment as described above.

[0131] The present invention also provides a pharmaceutical composition for administering to a mammalian subject to treat a thyroid - related disorder, which contains the fully human TSH receptor - blocking monoclonal antibody or its antigen - binding fragment as described above and a pharmaceutically acceptable carrier;

[0132] Wherein, the thyroid - related disorder is selected from: hyperthyroidism, thyroid - related ophthalmopathy (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin - induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema, etc.

[0133] Furthermore, the preparation, drug or pharmaceutical composition may further comprise physiologically compatible excipients, which include buffering agents, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc.

[0134] Furthermore, the preparation, drug or pharmaceutical composition can be made into injections, sterile powders for injection, tablets, pills, capsules, lozenges, spirits, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalants, or suppositories, etc. The preparations, drugs or pharmaceutical compositions of the above various dosage forms can all be prepared according to conventional methods in the pharmaceutical field.

[0135] Furthermore, the preparation, drug or pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal dropping, eye dropping, osmosis, absorption, physical or chemical mediated methods; or be introduced into the body after being mixed or encapsulated by other substances.

[0136] Preferably, the reagent or kit for detecting hyperthyroidism and thyroid-related ophthalmopathy contains the fully human TSH receptor-blocking monoclonal antibody.

[0137] Furthermore, 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.

[0138] Furthermore, the vector can be a plasmid, a virus or a fragment thereof, and various different types of vectors known to those skilled in the art.

[0139] Preferably, the host cell is CHO-K1 cell, etc.

[0140] Specifically, the composition may comprise a TSHR autoantibody with a determined concentration having TSH antagonist activity, and include a fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment according to the present invention.

[0141] Furthermore, the pharmaceutical composition is suitable for administration to humans; preferably, the pharmaceutical composition according to the present invention has no significant adverse effects on the immune system of the subject.

[0142] Furthermore, the pharmaceutical composition includes one or more additional thyroid-stimulating hormone receptor antagonists.

[0143] Furthermore, the pharmaceutical composition is used to treat a thyroid-related disorder in an injectable form.

[0144] Preferably, the pharmaceutical composition for treating Graves' ophthalmopathy is in the form of an intravenous injection preparation or an eye drop.

[0145] Preferably, the pharmaceutical composition for treating pretibial myxedema is in a form of topical administration.

[0146] Specifically, the pharmaceutical composition includes any antibody according to the present invention and any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical composition of the present invention include, but are not limited to, buffering substances (such as phosphates), glycine, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin), sorbic acid, potassium sorbate, water, sodium chloride, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, partial glyceride mixtures of saturated vegetable fatty acids, zinc salts, silica sols, cellulose-based substances, polyethylene glycols, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene block polymers, magnesium trisilicate, polyvinylpyrrolidone, polyethylene glycol and lanolin.

[0147] The dosage forms of the pharmaceutical composition include capsules, tablets, aqueous suspensions, solutions, rectal suppositories, enemas, ointments, lotions, creams, nasal sprays, inhalants. Preferably, they are solution and ointment preparations.

[0148] Specifically, the pharmaceutical composition can be in the form of a sterile injectable preparation, such as a sterile injectable oil suspension or aqueous suspension. Such suspensions can be made using suitable dispersing or wetting agents (such as Tween 80) and suspending agents according to techniques known in the art. The sterile injectable reagent can also be a sterile injectable solution or suspension made from a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable carriers and solvents that can be used are water, mannitol, Ringer's solution and isotonic sodium chloride solution. Additionally, sterile non-volatile oils are generally used as solvents or suspending media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids (such as oleic acid and its glyceride derivatives) can be used to prepare injectables. The above oil solutions or suspensions can also contain long-chain alcohol diluents or dispersing agents.

[0149] Specifically, the pharmaceutical composition can be administered by topical administration, by inhalation via spraying, orally, parenterally, by eye drops or eye ointment, orally, vaginally, rectally, nasally or via an implantable reservoir, etc. Preferably, it is oral administration or injection administration. The term "parenteral" as used in the present invention includes subcutaneous, intradermal, intrasynovial, intrasternal, intravenous, intramuscular, intralesional, intracranial, intra-articular and intrathecal injection or infusion techniques.

[0150] Specifically, the pharmaceutical composition can also be administered rectally in the form of suppositories. These compositions can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperature and will thus dissolve in the rectum to release the active ingredient. Such materials include, but are not limited to, beeswax, cocoa butter, and polyethylene glycol.

[0151] Specifically, the pharmaceutical composition can be administered orally in any orally acceptable dosage form, including, but not limited to, tablets, capsules, aqueous suspensions, and solutions. For oral tablets, common carriers include corn starch and lactose. Lubricants such as magnesium stearate are usually also added. For oral capsules, useful diluents include lactose and dry corn starch. When an aqueous suspension is administered orally, its active ingredient is combined with emulsifying and suspending agents. If desired, certain flavoring and / or sweetening and / or coloring agents can also be added.

[0152] Specifically, the pharmaceutical composition can be administered in the form of a nasal spray or an inhalant. These compositions are prepared according to techniques well known in the pharmaceutical formulation art and can be prepared as a saline solution using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other dispersants or solubilizing agents known in the art.

[0153] Specifically, when the desired treatment involves areas or organs easily accessible by topical administration, topical administration of the pharmaceutical composition of the present invention is particularly useful. For topical skin administration, the pharmaceutical composition should be formulated as a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical use of the compounds of the present invention include, but are not limited to, mineral oil, white petrolatum, propylene glycol, liquid petrolatum, emulsifying wax, polyoxyethylene polyoxypropylene compounds, and water. Alternatively, the pharmaceutical composition can be formulated as 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, polysorbate 60, cetyl esters wax, cetostearyl alcohol, sorbitan monostearate, benzyl alcohol, 2-octyldodecanol, and water. The pharmaceutical composition of the present invention can also be used for the lower intestine by rectal suppository formulation or in the form of a suitable enema. The present invention also includes topical transdermal patches.

[0154] The present invention also provides a method for preparing a fully human TSH receptor-blocking monoclonal antibody or an antigen-binding fragment thereof, and the preparation method specifically comprises the following steps:

[0155] First step: Sort plasma cells and memory B cells against TSHR from the peripheral blood of patients with relatively high TBAb activity, extract single-cell RNA and synthesize cDNA, and verify the amplification of heavy chain H, light chain λ, and light chain κ of the sorted single cells by nested PCR. Select single-cell clones with both heavy chain and light chain being positive for subsequent cloning.

[0156] Second step: Clone the BCR heavy chain and light chain of all single B cells into the heavy chain expression vector AbVec-IGHG1, λ light chain expression vector AbVec-hIgKappa, or λ light chain expression vector AbVec-hIgLambda by in vitro amplification with nested PCR.

[0157] Third step: After successfully obtaining the heavy chain and light chain recombinant plasmids, sequence and perform alignment analysis on the obtained candidate clones to clarify the nucleotide and amino acid sequence numbers of the obtained candidate antibodies; transfect the heavy chain and light chain expression plasmids to express monoclonal antibodies in vitro; further verify the antigen-binding ability and antibody blocking activity, and finally obtain an antibody combination that can specifically target the target antigen, namely the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment.

[0158] The present invention also provides a method for treating a thyroid-related disorder in a mammalian subject or in cells derived from the subject, the method comprising contacting the subject or the cells with the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment as described above.

[0159] Wherein, the thyroid-related disorder is selected from: hyperthyroidism, thyroid-related ophthalmopathy (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema, etc.

[0160] The present invention also provides a method for inhibiting the stimulation of the TSH receptor by thyroid-stimulating antibodies in the thyroid of a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment of the present invention;

[0161] Preferably, prevent the binding of thyroid-stimulating antibodies to the TSH receptor.

[0162] The present invention also provides a method for inhibiting the binding of thyroid-stimulating autoantibodies to the TSH receptor outside the thyroid in a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment as described above;

[0163] Specifically, the extra-thyroid TSH receptor is located in the retro-orbital tissue and / or pretibial tissue of the subject;

[0164] Preferably, the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment can block the binding of TSH receptor autoantibodies to the extra-thyroid TSH receptor.

[0165] The present invention also provides a method for treating thyroid cancer or metastatic thyroid cancer in a subject or in thyroid cells derived from a subject, the method comprising contacting the cancer cells with the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment as described above, with the aim of inhibiting constitutive thyroid-stimulating hormone receptor activity in the cells;

[0166] Preferably, the regrowth of the thyroid cancer cells is prevented or delayed.

[0167] The present invention also provides a method for treating thyroid hyperactivity caused by constitutive thyroid activity in a subject or in thyroid cells derived from a subject, the method comprising contacting the subject or the cells with the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment as described above, with the aim of inhibiting such thyroid hyperactivity.

[0168] The present invention also provides a method for identifying a molecule that can inhibit the binding of thyroid-stimulating antibodies to the TSH receptor, the method comprising providing at least one fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment as described above as a reference;

[0169] Preferably, a molecule that can prevent the binding of thyroid-stimulating antibodies to the TSH receptor is selected.

[0170] The present invention also provides a method for identifying a molecule that can inhibit the binding of thyroid-blocking antibodies to the TSH receptor, the method comprising providing at least one fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment as described above as a reference;

[0171] Preferably, a molecule that can prevent the binding of thyroid-blocking antibodies to the TSH receptor is selected.

[0172] Specifically, the thyroid-related disorders are selected from hyperthyroidism, thyroid-related ophthalmopathy (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema, etc.

[0173] Preferably, the subject treated in the above method is a human.

[0174] In the present invention, the preparation method includes the following steps: sorting plasma cells and memory single B cells that specifically recognize TSHR from the peripheral blood of patients with high titers of TSH receptor blocking antibodies (TSH-stimulating blocking antibody, TBAb) by flow cytometry, cloning the heavy and light chains of the antibody in vitro and recombinantly expressing them, and screening and verifying the antibody properties using hTSHR-CHO cells to obtain a blocking monoclonal antibody that specifically targets the human TSHR. Through the preparation method of the fully human 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.

[0175] Furthermore, the present invention verifies the antibody properties and evaluates the effects in vitro using hTSHR-CHO cells.

[0176] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment as described above for treating a thyroid-related disorder.

[0177] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment as described above in the preparation of a drug for treating a thyroid-related disorder.

[0178] The present invention also provides the application of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment as described above in the preparation of a reagent or kit for detecting TSH receptor antibodies.

[0179] The present invention also provides the application of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment as described above in the preparation of a reagent or kit or product for detecting hyperthyroidism and thyroid-related eye diseases.

[0180] The present invention also provides the application of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment, or the substance as described above, or the method as described above in the preparation of a preparation, drug or pharmaceutical composition for detecting TSH receptor antibodies, the preparation of a drug for treating hyperthyroidism and thyroid-related eye diseases, the preparation of a drug for inhibiting thyroid hyperplasia and / or thyroid hormone production, the preparation of a drug for TSH receptor blockade, and the preparation of a drug for antagonizing the activation effect of TSH on the TSH receptor.

[0181] Specifically, the thyroid-related disorders in the above method are selected from hyperthyroidism, thyroid-related eye diseases (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema, etc.

[0182] The method of the present invention, since it does not require immunizing animals, amplifies antibody genes directly from human single B cells, and amplifies the antibody genes of isolated single plasma cells or memory B cells, thereby obtaining a large number of naturally paired antibody heavy and light chain genes. Then, by expressing the paired antibody heavy and light chain genes, antibodies with antigen specificity and neutralizing activity are finally screened. The method of the present invention has the advantages of being fast, high-throughput, and requiring a small amount of cells. The fully human antibodies prepared retain rich gene diversity and natural pairing of heavy and light chain variable regions, and have great advantages. Compared with the traditional hybridoma antibody preparation technology, the method of the present invention can significantly shorten the experimental period. The traditional hybridoma antibody preparation technology generally 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.

[0183] The beneficial effects of the present invention also include: the fully human TSH receptor-blocking monoclonal antibody or its antigen-binding fragment of the present invention can effectively block the signal transduction after TSH binds to the receptor; inhibit the synthesis and secretion of thyroid hormones; significantly inhibit the expression and fibrosis of TSHR in orbital fibroblasts, the effector cells of thyroid-associated ophthalmopathy. It can be used to treat a series of diseases caused by hyperthyroidism such as thyroid ophthalmopathy, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema, etc., and has broad application prospects. Brief Description of the Drawings

[0184] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0185] Figure 1 This is for the flow cytometry technology of the present invention to sort plasma cells and memory single B cells that specifically recognize TSHR from peripheral blood. Using Biotin-AF647-labeled TSHR protein as a bait, the other antibodies used for sorting are: CD19-PacificBlue, IgM-PE, CD27-BV605, CD38-PE-Cy7. Finally, the TSHR-specific plasma cells and memory single B cells sorted are CD19 + IgM - CD27 + CD38 - TSHR + .

[0186] Figure 2This is the screening graph of the blocking activity of the anti-human TSHR monoclonal antibody of the present invention. The results shown are the mean ± standard deviation (n = 3). The dotted line value is 30%.

[0187] Figure 3 This is the result of the phylogenetic tree analysis of the anti-human TSHR monoclonal antibody sequence of the present invention. The percentage of the replicate trees in which the related taxa clustered together in the bootstrap test (1000 replicates) is shown below the branches.

[0188] Figure 4 This is the evaluation of the inhibitory effect of the SH1 antibody in the present invention on orbital fibroblasts. The horizontal line numbers represent the statistical differences (P values) of the antibody group compared with the control group (Control). A value less than or equal to 0.05 indicates a significant difference. Detailed implementation mode

[0189] Combined with the following specific embodiments and drawings, the present invention will be further described in detail. The processes, conditions, experimental methods, etc. for implementing the present invention are all general knowledge and common general knowledge in the art except for the specifically mentioned content below, and the present invention has no particularly restricted content.

[0190] Example 1

[0191] Single cell sorting: Collect the peripheral blood of volunteers with high TBAb titers, add the immunodensity gradient centrifugation human B cell enrichment mixture (STEMCELL, product number: 15024) to the blood sample, and use -1077 (Sigma, product number: 10771) for density gradient centrifugation to obtain B cells in peripheral blood. Sort and collect CD19 + IgM - CD27 + CD38 - TSHR + plasma cells and memory single B cells ( Figure 1 ).

[0192] Heavy and light chain variable region cloning: Use the SPRlselect nucleic acid fragment screening kit (Beckman Coulter, product number: B23317) to capture the RNA binding to single cells. According to SuperScript TMIV One-step RT-PCR System (Invitrogen, catalog number: 12594100) instruction manual was used to synthesize cDNA. Using the cDNA as a template, DreamTaq Green PCR 2X MasterMix (ThermoFisher, catalog number: K1081) was used to perform PCR amplification on the variable regions of the heavy and light chains of the antibody respectively. Subsequently, the amplified PCR products were detected by agarose gel electrophoresis. The bands with fragment sizes consistent with the expected values were excised and recovered, and the DNA fragments were purified using the QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704) and sent for sequencing. 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 to perform cloning PCR on the heavy and light chains. Subsequently, HiFi DNA Assembly Master Mix (NEB, catalog number: E2621L) was used for fragment ligation, and the obtained variable region sequences of the heavy and light chains were respectively cloned into the corresponding heavy and light chain expression vectors containing the constant regions of the heavy and light chains (NCBI GenBank accession numbers: FJ475055, FJ475056, FJ517647). After transformation, plating, and picking colonies, sequencing was performed to determine the final sequences.

[0193] Blocking antibody screening:

[0194] 1. Antigen binding ability screening: The heavy and light chain expression vectors of the antibody were co-transfected into 293T cells at a ratio of 1:1. After culturing at 37°C and 5% CO2 for 3 days, the culture supernatant was collected by centrifugation, and the titer of the TRAb antibody in the supernatant was detected according to the instruction manual of the Human Anti-Thyrotropin Receptor Antibody Enzyme-Linked Immunosorbent Assay Kit (Kelut, catalog number: ELK9540). The heavy and light chain combinations with binding ability lower than that of the blank control group were removed, and the remaining combinations were screened for blocking activity.

[0195] 2. Blocking activity screening: The heavy and light chain expression vectors of the antibody were co-transfected into 293T cells at a ratio of 1:1. After culturing at 37°C and 5% CO2 for 3 days, the culture supernatant was collected by centrifugation. Using 100 μL of the supernatant, 1 IU / L bTSH (Sigma) was added simultaneously, and after incubating hTSHR-CHO cells for 2 hours, the cell lysate was collected, and the change in the cAMP level in the cells was detected (R&D, catalog number: KGE002B).

[0196] Antibody expression and purification: The heavy and light chain expression vectors of the antibody were co-transfected into 293F cells at a ratio of 1:1; after culturing with shaking at 37 °C, 8% CO2, and 130 rpm for 5 days, the culture supernatant was collected by centrifugation, filtered through 0.45 μm, and purified by Protein A (GenScript, product number: L00210) affinity chromatography to obtain antibody protein with high purity; the antibody concentration was determined by Bradford protein concentration method (Beyotime, product number: P0006) and NanoDrop A280 method.

[0197] Evaluation of in vitro TSHR inhibitory effect: The purified monoclonal antibody was diluted according to different concentration gradients, 5 ng / ml of bTSH was added, and after co-incubating hTSHR-CHO cells for 2 hours, the cell lysate was collected to detect the change in the cAMP level of the cells. If the percentage of cAMP production inhibition was greater than 30%, it was considered that the monoclonal antibody had inhibitory activity. It was found that when the monoclonal antibody concentration was 1 μg / mL, it could effectively antagonize the activation of the TSH receptor by TSH and showed concentration dependence ( Figure 2 ). Among them, the percentage of cAMP production inhibition by SH1 reached 94% at 2 μg / mL. SH1 is the most inhibitory human-derived TSHR inhibitory antibody in this group of inhibitory antibodies.

[0198] Phylogenetic tree analysis: The phylogenetic analysis of the antibody was performed using MEGA 11 analysis software. The phylogenetic evolutionary tree was constructed by the neighbor-joining method, and the reliability of the evolutionary tree was evaluated by the Bootstrap value. The percentage of replicate trees in which the related taxa clustered together in the bootstrap test (1000 replicates) was shown below the branches, and the evolutionary distance was calculated using the Poisson correction method. Figure 3 Combined with the results of in vitro inhibitory activity determination for the unrooted optimal tree, antibody K1-70 is a reported TSHR inhibitory antibody, and the rest are antibodies obtained in this screening. Antibodies with closer evolutionary distances have more similar inhibitory activities. The results showed that the evolutionary distance between K1-70 and the most inhibitory antibody SH1 in the present invention was the farthest, indicating that the sequence differences between these two antibodies were the largest, and they were two TSHR inhibitory monoclonal antibodies with completely different sequences. And the TSHR inhibitory monoclonal antibody SH63 screened in the present invention with the closest evolutionary distance to K1-70 had an inhibitory activity of only 87% at an antibody concentration of 2 μg / mL, indicating that the inhibitory efficiency of K1-70 on TSHR was lower than that of the most inhibitory antibody SH1 screened in this study.

[0199] Evaluation of the inhibitory effect on orbital fibroblasts (OF cells): OF cells were cultured in a 10 cm dish containing DMEM with 10% FBS, and when they grew to 70%, they were subcultured to a 12-well plate and cultured for 16 hours with DMEM containing 1% FBS. After adding 50 ng / ml of the inhibitory antibody SH1, the culture was continued for 24 hours. The cells were collected to extract RNA, and RT-qPCR was performed to detect the expression level of fibrosis-related gene mRNA. The results showed that the TSHR inhibitory monoclonal antibody SH1 could significantly inhibit the expression of TSHR mRNA and the fibrosis marker collagen 1A1 (COL1A1) mRNA ( Figure 4 ).

[0200] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0201] As used in the present invention, the terms "include" and "comprising" are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0202] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0203] 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 group of fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof, characterized in that: The fully human TSH receptor blocking monoclonal antibody or its antigen binding fragment binds to the TSH receptor to block the binding of TSH to the TSH receptor; the fully human TSH receptor blocking monoclonal antibody or its antigen binding fragment comprises a heavy chain variable region and a light chain variable region; Wherein, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, the amino acid sequence of the H-CDR1 is selected from one of SEQ ID NOs: 30 to 33; the amino acid sequence of the H-CDR2 is selected from one of SEQ ID NOs: 34 to 36; the amino acid sequence of the H-CDR3 is selected from one of SEQ ID NOs: 37 to 40; The light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, the amino acid sequence of the L-CDR1 is selected from one of SEQ ID NOs: 41 to 42; the amino acid sequence of the L-CDR2 is selected from one of SEQ ID NOs: 43 to 45; the amino acid sequence of the L-CDR3 is selected from one of SEQ ID NOs: 46 to 48.

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, 13 or has at least 85% sequence identity with one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13; The amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or has at least 85% sequence identity with one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14.

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, and 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 fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof is a TSH antagonist; and / or is an antagonist of thyroid stimulating antibodies.

5. A substance as described in any one of the following, characterized in that The substances include: (1) A preparation, a drug or a pharmaceutical composition, comprising the fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof as claimed in claim 1 or 2; (2) A reagent or a kit, comprising the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2; (3) A nucleotide encoding the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the nucleotide sequence comprises: (a) the nucleotide sequence encoding the heavy chain variable region is shown in one of SEQ ID NOs: 15, 17, 19, 21, 23, 25, and 27; the nucleotide sequence encoding the light chain variable region is shown in one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, and 28; (b) a nucleotide sequence having at least 85% sequence identity to one of SEQ ID NO: 15 to SEQ ID NO: 28; (c) a nucleotide sequence after one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO: 15 to SEQ ID NO: 28; (d) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a), (b) or (c) above or its full-length complement; or, (e) a nucleotide sequence that is different from the nucleotide sequences of (a), (b), (c), and (d) above due to the degeneracy of the genetic code; Wherein, the nucleotide sequence of the nucleotide or a part thereof encodes antibody V H Domain and antibody V L A domain or a CDR selected from SEQ ID NO: 30 to SEQ ID NO: 48; (4) A vector comprising the nucleotide described in (3); (5) A host cell, comprising the nucleotide described in (3) and / or the vector described in (4); (6) A cell, wherein the cell is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, and / or the nucleotide according to (3), and / or the vector according to (4); and / or, An isolated cell expressing the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2; and / or, An isolated cell secreting the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2; (7) A composition comprising a determined concentration of TSH receptor autoantibodies and a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2; (8) A pharmaceutical composition for administration to a mammalian subject to treat a thyroid-related disorder, the pharmaceutical composition comprising the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 and a pharmaceutically acceptable carrier; Wherein, the thyroid-related disease is selected from: hyperthyroidism, thyroid-related eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis and pretibial myxedema.

6. The substance according to claim 5, characterized in that The pharmaceutical composition is suitable for administration to humans.

7. The substance according to claim 5, characterized in that The pharmaceutical composition includes one or more additional thyroid stimulating hormone receptor antagonists.

8. The substance according to claim 5, characterized in that The pharmaceutical composition comprises the 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 according to claim 5, characterized in that The administration of the pharmaceutical composition includes oral administration, parenteral administration, administration by spray inhalation, topical administration, administration by eye drops or eye ointment, rectal administration, nasal administration, oral administration, vaginal administration, and administration via an implanted reservoir.

10. The substance according to claim 5, characterized in that The pharmaceutical composition may be in the form of capsules, tablets, aqueous suspensions, solutions, rectal suppositories, enemas, ointments, lotions, creams, nasal sprays, and inhalants.

11. The substance according to claim 5, characterized in that The pharmaceutical composition is used to treat a thyroid-related disorder in an injectable form.

12. The substance according to claim 5, characterized in that The pharmaceutical composition is used for treating pretibial myxedema in a topical administration form; and / or, 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 method for preparing a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof, the preparation method specifically comprising the following steps: In the first step, 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 and light chains were selected for subsequent cloning; In the second step, 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; In the third step, after the heavy chain and light chain recombinant plasmids are successfully obtained, 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; 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 or its antigen binding fragment, 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 antigen-binding fragment thereof according to claim 1 or 2; (3) A method for inhibiting thyroid stimulating antibodies from stimulating TSH receptors in the thyroid gland of a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2; (4) A method for inhibiting the binding of thyroid stimulating autoantibodies to extrathyroidal TSH receptors in a mammalian subject, the method comprising contacting the subject with a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2; (5) A method for treating thyroid cancer or metastatic thyroid cancer in a subject or in thyroid cells derived from a subject, the method comprising contacting the cancer cells with a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2, with the purpose of inhibiting constitutive thyroid stimulating hormone receptor activity in the cells; (6) A method for treating thyroid hyperactivity caused by constitutive thyroid activity in a subject or in thyroid cells derived from the subject, characterized in that the method comprises contacting the subject or the cell with a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2, in order to inhibit such thyroid hyperactivity; (7) A method for identifying a molecule that can inhibit the binding of a thyroid stimulating antibody to a TSH receptor, the method comprising providing at least one fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2 as a reference; (8) A method for identifying a molecule that can inhibit the binding of a thyroid blocking antibody to a TSH receptor, the method comprising providing at least one fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2 as a reference.

14. The method according to claim 13, characterized in that The thyroid-related disorder is selected from the group consisting of hyperthyroidism, thyroid-related eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema.

15. The method according to claim 13, characterized in that The subject is a human.

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

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

18. The method according to claim 13, characterized in that In method (5), the method prevents or delays the regrowth of thyroid cancer cells.

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

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

21. Any of the following applications, characterized in that: The applications include: (1) Use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 for treating a thyroid-related disease; (2) Use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a medicament for treating a thyroid-related disease; (3) Use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a reagent or kit for detecting TSH receptor antibodies; (4) Use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 in the preparation of a reagent, kit or product for detecting hyperthyroidism or thyroid-related eye diseases; (5) Use of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, or the substance according to claim 5, or the method according to claim 13 in the preparation of a preparation, a drug or a pharmaceutical composition for detecting TSH receptor antibodies, the preparation of a drug for treating hyperthyroidism and thyroid-related eye diseases, the preparation of a drug for inhibiting thyroid hyperplasia and / or thyroid hormone production, the preparation of a TSH receptor blocking drug, and the preparation of a drug for antagonizing the activation effect of TSH on TSH receptors.

22. The use according to claim 21, characterized in that The thyroid-related disorder is selected from the group consisting of hyperthyroidism, thyroid-related eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema.

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